System of components for preparing oligonucleotides

ABSTRACT

Interative, preferably computer based iterative processes for generating synthetic compounds with desired physical, chemical and/or bioactive properties, i.e., active compounds, are provided. During iterations of the processes, a target nucleic acid sequence is provided or selected, and a library of candidate nucleobase sequences is generated in silico according to defined criteria. A “virtual” oligonucleotide chemistry is chosen and a library of virtual oligonucleotide compounds having the selected nucleobase sequences is generated. These virtual compounds are reviewed and compounds predicted to have particular properties are selected. The selected compounds are robotically synthesized and are preferably robotically assayed for a desired physical, chemical or biological activity. Active compounds are thus generated and, at the same time, preferred sequences and regions of the target nucleic acid that are amenable to oligonucleotide or sequence-based modulation are identified.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the generation ofsynthetic compounds having defined physical, chemical or bioactiveproperties. More particularly, the present invention relates to theautomated generation of oligonucleotide compounds targeted to a givennucleic acid sequence via computer-based, iterative robotic synthesis ofsynthetic oligonucleotide compounds and robotic or robot-assistedanalysis of the activities of such compounds. Information gathered fromassays of such compounds is used to identify nucleic acid sequences thatare tractable to a variety of nucleotide sequence-based technologies,for example, antisense drug discovery and target validation.

BACKGROUND OF THE INVENTION

[0002] 1. Oligonucleotide Technology

[0003] Synthetic oligonucleotides of complementarity to targets areknown to hybridize with particular, target nucleic acids. In oneexample, compounds complementary to the ‘sense’ strand of nucleic acidsthat encode polypeptides, are referred to as “antisenseoligonucleotides.” A subset of such compounds may be capable ofmodulating the expression of target nucleic acid in vivo; such syntheticcompounds are described herein as “active oligonucleotide compounds.”

[0004] Oligonucleotide compounds are also commonly used in vitro asresearch reagents and diagnostic aids, and in vivo as therapeutic andbioactive agents. Oligonucleotide compounds can exert their effect by avariety of means. One such means is the antisense-mediated use of anendogenous nuclease, such as RNase H in eukaryotes or RNase P inprokaryotes, to the target nucleic acid (Chiang et al., J. Biol. Chem.,1991, 266, 18162; Forster et al., Science, 1990, 249, 783). Anothermeans involves covalently linking of a synthetic moiety having nucleaseactivity to an oligonucleotide having an antisense sequence. This doesnot rely upon recruitment of an endogenous nuclease to modulate targetactivity. Synthetic moieties having nuclease activity include, but arenot limited to, enzymatic RNAs, lanthanide ion complexes, and otherreactions species. (Haseloff et al., Nature, 1988, 334, 585; Baker etal., J. Am. Chem. Soc., 1997, 119, 8749).

[0005] Despite the advances made in utilizing antisense technology todate, it is still common to identify sequences amenable to antisensetechnologies through an empirical approach (Szoka, Nature Biotechnology,1997, 15, 509). Accordingly, the need exists for systems and methods forefficiently and effectively identifying target nucleotide sequences thatare suitable for antisense modulation. The present disclosure answersthis need by providing systems and methods for automatically identifyingsuch sequences via in silico, robotic or other automated means.

[0006] 2. Identification of Active Oligonucleotide Compounds

[0007] Traditionally, new chemical entities with useful properties aregenerated by (1) identifying a chemical compound (called a ‘leadcompound’) with some desirable property or activity, (2) creatingvariants of the lead compound, and (3) evaluating the property andactivity of such variant compounds. The process has been called ‘SAR’,i.e., structure activity relationship. Although ‘SAR’ and itshandmaiden, rational drug design, has been utilized with some degree ofsuccess, there are a number of limitations to these approaches to leadcompound generation, particularly as it pertains to the discovery ofbioactive oligonucleotide compounds. In attempting to use SAR witholigonucleotides, it has been recognized that RNA structure can inhibitduplex formation with antisense compounds, so much so that “moving” thetarget nucleotide sequence even a few bases can drastically decrease theactivity of such compounds (Lima et al., Biochemistry, 1992, 31, 12055).

[0008] Heretofore, the search for lead antisense compounds has beenlimited to the manual synthesis and analysis of such compounds.Consequently, a fundamental limitation of the conventional approach isits dependence upon the availability, number and cost of antisensecompounds produced by manual, or at best semi-automated, means.Moreover, the assaying of such compounds has traditionally beenperformed by tedious manual techniques. Thus, the traditional approachto generating active antisense compounds is limited by the relativelyhigh cost and long time required to synthesize and screen a relativelysmall number of candidate antisense compounds.

[0009] Accordingly, the need exists for systems and methods forefficiently and effectively generating new active antisense and otherolgonucleotide compounds targeted to specific nucleic acid sequences.The present disclosure answers this need by providing systems andmethods for automatically generating active antisense compounds viarobotic and other automated means.

[0010] 3. Gene Function Analysis

[0011] Efforts such as the Human Genome Project are making an enormousamount of nucleotide sequence information available in a variety offorms, e.g., genomic sequences, cDNAs, expressed sequence tags (ESTs)and the like. This explosion of information has led one commentator tostate that ‘genome scientists are producing more genes than they can puta function to’ (Kahn, Science, 1995, 270, 369). Although some approachesto this problem have been suggested, no solution has yet emerged. Forexample, methods of looking at gene expression in different diseasestates or stages of development only provide, at best, an associationbetween a gene and a disease or stage of development (Nowak, Science,1995, 270, 368). Another approach, looking at the proteins encoded bygenes, is developing but ‘this approach is more complex and bigobstacles remain’ (Kahn, Science, 1995, 270, 369). Furthermore, neitherof these approaches allows one to directly utilize nucleotide sequenceinformation to perform gene function analysis.

[0012] In contrast, antisense technology does allow for the directutilization of nucleotide sequence information for gene functionanalysis. Once a target nucleic acid sequence has been selected,antisense sequences hybridizable to the sequence can be generated usingtechniques known in the art. Typically, a large number of candidateantisense oligonucleotides (ASOs) are synthesized having sequences thatare more-or-less randomly spaced across the length of the target nucleicacid sequence (e.g., a ‘gene walk’) and their ability to modulate theexpression of the target nucleic acid is assayed. Cells or animals canthen be treated with one or more active antisense oligonucleotides, andthe resulting effects determined in order to determine the function(s)of the target gene. Although the practicality and value of thisempirical approach to developing active antisense compounds has beenacknowledged in the art, it has also been stated that this approach ‘isbeyond the means of most laboratories and is not feasible when a newgene sequence is identified, but whose function and therapeuticpotential are unknown’ (Szoka, Nature Biotechnology, 1997, 15, 509).

[0013] Accordingly, the need exists for systems and methods forefficiently and effectively determining the function of a gene that isuncharacterized except that its nucleotide sequence, or a portionthereof, is known. The present disclosure answers this need by providingsystems and methods for automatically generating active antisensecompounds to a target nucleotide sequence via robotic means. Such activeantisense compounds are contacted with cells, cell-free extracts,tissues or animals capable of expressing the gene of interest andsubsequent biochemical or biological parameters are measured. Theresults are compared to those obtained from a control cell culture,cell-free extract, tissue or animal which has not been contacted with anactive antisense compound in order to determine the function of the geneof interest.

[0014] 4. Target Validation

[0015] Determining the nucleotide sequence of a gene is no longer an endunto itself; rather, it is ‘merely a means to an end. The critical nextstep is to validate the gene and its [gene] product as a potential drugtarget’ (Glasser, Genetic Engineering News, 1997, 17, 1). This process,i.e., confirming that modulation of a gene that is suspected of beinginvolved in a disease or disorder actually results in an effect that isconsistent with a causal relationship between the gene and the diseaseor disorder, is known as target validation.

[0016] Efforts such as the Human Genome Project are yielding a vastnumber of complete or partial nucleotide sequences, many of which mightcorrespond to or encode targets useful for new drug discovery efforts.The challenge represented by this plethora of information is how to usesuch nucleotide sequences to identify and rank valid targets for drugdiscovery. Antisense technology provides one means by which this mightbe accomplished; however, the many manual, labor-intensive and costlysteps involved in traditional methods of developing active antisensecompounds has limited their use in target validation (Szoka, NatureBiotechnology, 1997, 15, 509). Nevertheless, the great targetspecificity that is characteristic of antisense compounds makes themideal choices for target validation, especially when the functionalroles of proteins that are highly related are being investigated (Albertet al., Trends in Pharm. Sci., 1994, 15, 250).

[0017] Accordingly, the need exists for systems and methods fordeveloping compounds efficiently and effectively that modulate a gene,wherein such compounds can be directly developed from nucleotidesequence information. Such compounds are needed to confirm thatmodulation of a gene that is thought to be involved in a disease ordisorder will in fact cause an in vitro or in vivo effect indicative ofthe origin, development, spread or growth of the disease or disorder.

[0018] The present disclosure answers this need by providing systems andmethods for automatically generating active oligonucleotide and othercompounds, especially antisense compounds, to a target nucleotidesequence via robotic or other automated means. Such active compounds arecontacted with a cell culture, cell-free extract, tissue or animalcapable of expressing the gene of interest, and subsequent biochemicalor biological parameters indicative of the origin, development, spreador growth of the disease or disorder are measured. These results arecompared to those obtained with a control cell system, cell-freeextract, tissue or animal which has not been contacted with an activeantisense compound in order to determine whether or not modulation ofthe gene of interest will have a therapeutic benefit or not. Theresulting active antisense compounds may be used as positive controlswhen other, non antisense-based agents directed to the same targetnucleic acid, or to its gene product, are screened.

[0019] It should be noted that embodiments of the invention drawn togene function analysis and target validation have parameters that areshared with other embodiments of the invention, but also have uniqueparameters. For example, antisense drug discovery naturally requiresthat the toxicity of the antisense compounds be manageable, whereas, forgene function analysis or target validation, overt toxicity resultingfrom the antisense compounds is acceptable unless it interferes with theassay being used to evaluate the effects of treatment with suchcompounds.

[0020] U.S. Pat. No. 5,563,036 to Peterson et al. describes systems andmethods of screening for compounds that inhibit the binding of atranscription factor to a nucleic acid. In a preferred embodiment, anassay portion of the process is stated to be performed by a computercontrolled robot.

[0021] U.S. Pat. No. 5,708,158 to Hoey describes systems and methods foridentifying pharmacological agents stated to be useful for diagnosing ortreating a disease associated with a gene the expression of which ismodulated by a human nuclear factor of activated T cells. The methodsare stated to be particularly suited to high-thoughput screening whereinone or more steps of the process are performed by a computer controlledrobot.

[0022] U.S. Pat. Nos. 5,693,463 and 5,716,780 to Edwards et al. describesystems and methods for identifying non-oligonucleotide molecules thatspecifically bind to a DNA molecule based on their ability to competewith a DNA-binding protein that recognizes the DNA molecule.

SUMMARY OF THE INVENTION

[0023] The present invention is directed to automated systems andmethods for generating active oligonucleotide compounds, i.e., thosehaving desired physical, chemical and/or biological properties. Thepresent invention is also directed to oligonucleotide-sensitive targetsequences identified, by the systems and methods. For purposes ofillustration, the present invention is described herein with respect tothe production of antisense oligonucleotides; however, the presentinvention is not limited to this embodiment.

[0024] The present invention is directed to iterative processes forgenerating new chemical compounds with prescribed sets of physical,chemical and/or biological properties, and to systems for implementingthese processes. During each iteration of a process as contemplatedherein, a target nucleic acid sequence is provided or selected, and alibrary of (candidate) nucleobase sequences is generated in silico (thatis in a computer manipulatible and reliable form) according to definedcriteria a virtual oligonucleotide chemistry is chosen. A library ofvirtual oligonucleotide compounds having the desired nucleobasesequences is generated. These virtual compounds are reviewed andcompounds predicted to have particular desired properties are selected.The selected compounds are synthesized, preferably in a robotic,batchwise manner; and then they are robotically assayed for a desiredphysical, chemical or biological activity in order to identify compoundswith the desired properties. Active compounds are, thus, generated and,at the same time, preferred sequences and regions of the target nucleicacid that are amenable to modulation are identified.

[0025] In subsequent iterations of the process, second libraries ofcandidate nucleobase sequences are generated and/or selected to giverise to a second virtual oligonucleotide library. Through multipleiterations of the process, a library of target nucleic acid sequencesthat are tractable to oligonucleotide technologies are identified. Suchmodulation includes, but is not limited to, antisense technology, genefunction analysis and target validation.

[0026] Further features and advantages of the present invention, as wellas the structure and operation of various embodiments of the presentinvention, are described in detail below with reference to theaccompanying drawings. In the drawings, like reference numbers indicateidentical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be described with reference to theaccompanying drawings, wherein:

[0028]FIGS. 1 and 2 are a flow diagram of one method according to thepresent invention depicting the overall flow of data and materials amongvarious elements of the invention.

[0029]FIG. 3 is a flow diagram depicting the flow of data and materialsamong elements of step 200 of FIG. 1.

[0030]FIGS. 4 and 5 are a flow diagram depicting the flow of data andmaterials among elements of step 300 of FIG. 1.

[0031]FIG. 6 is a flow diagram depicting the flow of data and materialsamong elements of step 306 of FIG. 4.

[0032]FIG. 7 is another flow diagram depicting the flow of data andmaterials among elements of step 306 of FIG. 4.

[0033]FIG. 8 is a another flow diagram depicting the flow of data andmaterials among elements of step 306 of FIG. 4.

[0034]FIG. 9 is a flow diagram depicting the flow of data and materialsamong elements of step 350 of FIG. 5.

[0035]FIGS. 10 and 11 are flow diagrams depicting a logical analysis ofdata and materials among elements of step 400 of FIG. 1.

[0036]FIG. 12 is a flow diagram depicting the flow of data and materialsamong the elements of step 400 of FIG. 1.

[0037]FIGS. 13 and 14 are flow diagrams depicting the flow of data andmaterials among elements of step 500 of FIG. 1.

[0038]FIG. 15 is a flow diagram depicting the flow of data and materialsamong elements of step 600 of FIG. 1.

[0039]FIG. 16 is a flow diagram depicting the flow of data and materialsamong elements of step 700 of FIG. 1.

[0040]FIG. 17 is a flow diagram depicting the flow of data and materialsamong the elements of step 1100 of FIG. 2.

[0041]FIG. 18 is a block diagram showing the interconnecting of certaindevices utilized in conjunction with a preferred method of theinvention;

[0042]FIG. 19 is a flow diagram showing a representation of data storagein a relational database utilized in conjunction with one method of theinvention;

[0043]FIG. 20 is a flow diagram depicting the flow of date and materialsin effecting a preferred embodiment of the invention as set forth inExample 14;

[0044]FIG. 21 is a flow diagram depicting the depicting the flow of dateand materials in effecting a preferred embodiment of the invention asset forth in Example 15;

[0045]FIG. 22 is a flow diagram depicting the depicting the flow of dateand materials in effecting a preferred embodiment of the invention asset forth in Example 2;

[0046]FIG. 23 is a pictorial elevation view of a preferred apparatusused to robotically synthesize oligonucleotides; and

[0047]FIG. 24 is a pictorial plan view of an apparatus used torobotically synthesize oligonucleotides.

[0048] Certain preferred methods of this invention are now describedwith reference to the flow diagram of FIGS. 1 and 2.

[0049] 1. Target Nucleic Acid Selection.

[0050] The target selection process, process step 100, provides a targetnucleotide sequence that is used to help guide subsequent steps of theprocess. It is generally desired to modulate the expression of thetarget nucleic acid for any of a variety of purposes, such as, e.g.,drug discovery, target validation and/or gene function analysis.

[0051] One of the primary objectives of the target selection process,step 100, is to identify molecular targets that represent significanttherapeutic opportunities, provide new medicines to the medicalcommunity to fill therapeutic voids or improve upon existing therapies,to provide new and efficacious means of drug discovery and to determinethe function of genes that are uncharacterized except for nucleotidesequence. To meet these objectives, genes are classified based uponspecific sets of selection criteria.

[0052] One such set of selection criteria concerns the quantity andquality of target nucleotide sequence. There must be sufficient targetnucleic acid sequence information available for oligonucleotide design.Moreover, such information must be of sufficient quality to give rise toan acceptable level of confidence in the data to perform the methodsdescribed herein. Thus, the data must not containing too many missing orincorrect base entries. In the case of a target sequence that encodes apolypeptide, such errors can be detected by virtually translating allthree reading frames of the sense strand of the target sequence andconfirming the presence of a continuous polypeptide sequence havingpredictable attributes, e.g., encoding a polypeptide of known size, orencoding a polypeptide that is about the same length as a homologousprotein. In any event, only a very high frequency of sequence errorswill frustrate the methods of the invention; most oligonucleotides tothe target sequence will avoid such errors unless such errors occurfrequently throughout the entire target sequence.

[0053] Another preferred criterion is that appropriate culturable celllines or other source of reproducible genetic expression should beavailable. Such cell lines express, or can be induced to express, thegene comprising the target nucleic acid sequence. The oligonucleotidecompounds generated by the process of the invention are assayed usingsuch cell lines and, if such assaying is performed robotically, the cellline is preferably tractable to robotic manipulation such as by growthin 96 well plates. Those skilled in the art will recognize that if anappropriate cell line does not exist, it will nevertheless be possibleto construct an appropriate cell line. For example, a cell line can betransfected with an expression vector comprising the target gene inorder to generate an appropriate cell line for assay purposes.

[0054] For gene function analysis, it is I-ossible to operate upon agenetic system having a lack of information regarding, or incompletecharacterization of, the biological function(s) of the target nucleicacid or its gene product(s). This is a powerful agent of the invention.A target nucleic acid for gene function analysis might be absolutelyuncharacterized, or might be thought to have a function based on minimaldata or homology to another gene. By application of the process of theinvention to such a target, active compounds that modulate theexpression of the gene can be developed and applied to cells. Theresulting cellular, biochemical or molecular biological responses areobserved, and this information is used by those skilled in the art toelucidate the function of the target gene.

[0055] For target validation and drug discovery, another selectioncriterion is disease association. Candidate target genes are placed intoone of several broad categories of known or deduced disease association.Level 1 Targets are target nucleic acids for which there is a strongcorrelation with disease. This correlation can come from multiplescientific disciplines including, but not limited to, epidemiology,wherein frequencies of gene abnormalities are associated with diseaseincidence; molecular biology, wherein gene expression and function areassociated with cellular events correlated with a disease; andbiochemistry, wherein the in vitro activities of a gene product areassociated with disease parameters. Because there is a strongtherapeutic rationale for focusing on Level 1 Targets, these targets aremost preferred for drug discovery and/or target validation.

[0056] Level 2 Targets are nucleic acid targets for which the combinedepidemiological, molecular biological, and/or biochemical correlationwith disease is not so clear as for Level 1. Level 3 Targets are targetsfor which there is little or no data to directly link the target with adisease process, but there is indirect evidence for such a link, i.e.,homology with a Level 1 or Level 2 target nucleic acid sequence or withthe gene product thereof. In order not to prejudice the target selectionprocess, and to ensure that the maximum number of nucleic acids actuallyinvolved in the causation, potentiation, aggravation, spread,continuance or after-effects of disease states are investigated, it ispreferred to examine a balanced mix of Level 1, 2 and 3 target nucleicacids.

[0057] In order to carry out drug discovery, experimental systems andreagents shall be available in order for one to evaluate the therapeuticpotential of active compounds generated by the process of the invention.Such systems may be operable in vitro (e.g., in vitro models ofcell:cell association) or in vivo (e.g., animal models of diseasestates). It is also desirable, but not obligatory, to have availableanimal model systems which can be used to evaluate drug pharmacology.

[0058] Candidate targets nucleic acids can also classified by biologicalprocesses. For example, programmed cell death (‘apoptosis’) has recentlyemerged as an important biological process that is perturbed in a widevariety of diseases. Accordingly, nucleic acids that encode factors thatplay a role in the apoptotic process are identified as candidatetargets. Similarly, potential target nucleic acids can be classified asbeing involved in inflammation, autoimmune disorders, cancer, or otherpathological or dysfunctional processes.

[0059] Moreover, genes can often be grouped into families based onsequence homology and biological function. Individual family members canact redundantly, or can provide specificity through diversity ofinteractions with downstream effectors, or through expression beingrestricted to specific cell types. When one member of a gene family isassociated with a disease process then the rationale for targeting othermembers of the same family is reasonably strong. Therefore, members ofsuch gene families are preferred target nucleic acids to which themethods and systems of the invention may be applied. Indeed, the potentspecificity of antisense compounds for different gene family membersmakes the invention particularly suited for such targets (Albert et al.,Trends Pharm. Sci., 1994, 15, 250). Those skilled in the art willrecognize that a partial or complete nucleotide sequence of such familymembers can be obtained using the polymerase chain reaction (PCR) and‘universal’ primers, i.e., primers designed to be common to all membersof a given gene family.

[0060] PCR products generated from universal primers can be cloned andsequenced or directly sequenced using techniques known in the art. Thus,although nucleotide sequences from cloned DNAs, or from complementaryDNAs (cDNAs) derived from mRNAs, may be used in the process of theinvention, there is no requirement that the target nucleotide sequencebe isolated from a cloned nucleic acid. Any nucleotide sequence, nomatter how determined, of any nucleic acid, isolated or prepared in anyfashion, may be used as a target nucleic acid in the process of theinvention.

[0061] Furthermore, although polypeptide-encoding nucleic acids providethe target nucleotide sequences in one embodiment of the invention,other nucleic acids may be targeted as well. Thus, for example, thenucleotide sequences of structural or enzymatic RNAs may be utilized fordrug discovery and/or target validation when such RNAs are associatedwith a disease state, or for gene function analysis when theirbiological role is not known.

[0062] 2. Assembly of Target Nucleotide Sequence.

[0063]FIG. 3 is a block diagram detailing the steps of the targetnucleotide sequence assembly process, process step 200 in acccordancewith one embodiment of the invention. The oligonucleotide designprocess, process step 300, is facilitated by the availability ofaccurate target sequence information. Because of limitations ofautomated genome sequencing technology, gene sequences are oftenaccumulated in fragments. Further, because individual genes are oftenbeing sequenced by independent laboratories using different sequencingstrategies, sequence information corresponding to different fragments isoften deposited in different databases. The target nucleic acid assemblyprocess take advantage of computerized homology search algorithms andsequence fragment assembly algorithms to search available databases forrelated sequence information and incorporate available sequenceinformation into the best possible representation of the target nucleicacid molecule, for example a RNA transcript. This representation is thenused to design oligonucleotides, process step 300, which can be testedfor biological activity in process step 700.

[0064] In the case of genes directing the synthesis of multipletranscripts, i.e. by alternative splicing, each distinct transcript is aunique target nucleic acid for purposes of step 300. In one embodimentof the invention, if active compounds specific for a given transcriptisoform are desired, the target nucleotide sequence is limited to thosesequences that are unique to that transcript isoform. In anotherembodiment of the invention, if it is desired to modulate two or moretranscript isoforms in concert, the target nucleotide sequence islimited to sequences that are shared between the two or moretranscripts.

[0065] In the case of a polypeptide-encoding nucleic acid, it isgenerally preferred that full-length cDNA be used in the oligonucleotidedesign process step 300 (with full-length cDNA being defined a readingfrom the 5′ cap to the poly A tail). Although full-length cDNA ispreferred, it is possible to design oligonucleotides using partialsequence information. Therefore it is not necessary for the assemblyprocess to generate a complete cDNA sequence. Further in some cases itmay be desirable to design oligonucleotides targeting introns. In thiscase the process can be used to identify individual introns at processstep 220.

[0066] The process can be initiated by entering initial sequenceinformation on a selected molecular target at process step 205. In thecase of a polypeptide-encoding nucleic acid, the full-length cDNAsequence is generally preferred for use in oligonucleotide designstrategies at process step 300. The first step is to determine if theinitial sequence information represents the full-length cDNA, decisionstep 210. In the case where the full-length cDNA sequence is availablethe process advances directly to the oligonucleotide design step 300.When the full-length cDNA sequence is not available, databases aresearched at process step 212 for additional sequence information.

[0067] The algorithm preferably used in process steps 212 and 230 isBLAST (Altschul, et al., J. Mol. Biol., 1990, 215, 403), or ‘GappedBLAST’ (Altschul et al., Nucl. Acids Res., 1997, 25, 3389). These aredatabase search tools based on sequence homology used to identifyrelated sequences in a sequence database. The BLAST search parametersare set to only identify closely related sequences. Some preferreddatabases searched by BLAST are a combination of public domain andproprietary databases. The databases, their contents, and sources arelisted in Table 1. TABLE 1 Database Sources of Target Sequences DatabaseContents Source NR All non-redundant National Center for GenBank, EMBL,DDBJ Biotechnology Information and PDB sequences at the NationalInstitutes of Health Month All new or revised National Center forGenBank, EMBL, DDBJ Biotechnology Information and PDB sequences at theNational released in the last Institutes of Health 30 days DbestNon-redundant National Center for database of GenBank, BiotechnologyInformation EMBL, DDBJ and EST at the National divisions Institutes ofHealth Dbsts Non-redundant National Center for database of GenBank,Biotechnology Information EMBL, DDBJ and STS at the National divisionsInstitutes of Health Htgs High throughput National Center for genomicsequences Biotechnology Information at the National Institutes of Health

[0068] When genomic sequence information is available at decision step215, introns are removed and exons are assembled into continuoussequence representing the cDNA sequence in process step 220. Exonassembly occurs using the Phragment Assembly Program ‘Phrap’ (CopyrightUniversity of Washington Genome Center, Seattle, Wash.). The Phrapalgorithm analyzes sets of overlapping sequences and assembles them intoone continuous sequence referred to as a ‘contig’. The resulting contigis preferably used to search databases for additional sequenceinformation at process step 230. When genomic information is notavailable, the results of process step 212 are analyzed for individualexons at decision step 225. Exons are frequently recorded individuallyin databases. If multiple complete exons are identified, they arepreferably assembled into a contig using Phrap at process step 250. Ifmultiple complete exons are not identified at decision step 225, thensequences can be analyzed for partial sequence information in decisionstep 228. ESTs identified in the database dbEST are examples of suchpartial sequence information. If additional partial information is notfound, then the process is advanced to process step 230 at decision step228. If partial sequence information is found in process 212 then thatinformation is advanced to process step 230 via decision step 228.

[0069] Process step 230, decision step 240, decision step 260 andprocess step 250 define a loop designed to extend iteratively the amountof sequence information available for targeting. At the end of eachiteration of this loop, the results are analyzed in decision steps 240and 260. If no new information is found then the process advances atdecision step 240 to process step 300. If there is an unexpectedly largeamount of sequence information identified, then the process ispreferably cycled back one iteration and that sequence is advanced atdecision step 240 to process step 300. If a small amount of new sequenceinformation is identified, then the loop is iterated such as by takingthe 100 most 5-prime (5′) and 100 most 3-prime (3′) bases and interatingthem through the BLAST homology search at process step 230. New sequenceinformation is added to the existing contig at process step 250.

[0070] This loop is iterated until either no new sequence information isidentified at decision step 240, or an unexpectedly large amount of newinformation is found at decision step 260, suggesting that the processmoved outside the boundary of the gene into repetitive genomic sequence.In either of these cases, iteration of this loop is preferably stoppedand the process advanced to the oligonucleotide design at process step300.

[0071] 3. In Silico Generation of a Set of Nucleobase Sequences andVirtual Oligonucleotides.

[0072] For the following steps 300 and 400, they may be performed in theorder described below, i.e., step 300 before step 400, or, in analternative embodiment of the invention, step 400 before step 300. Inthis alternate embodiment, each oligonucleotide chemistry is firstassigned to each oligonucleotide sequence. Then, each combination ofoligonucleotide chemistry and sequence is evaluated according to theparameters of step 300. This embodiment has the desirable feature oftaking into account the effect of alternative oligonucleotidechemistries on such parameters. For example, substitution of 5-methylcytosine (5 MeC or m5 c) for cytosine in an antisense compound mayenhance the stability of a duplex formed between that compound and itstarget nucleic acid. Other oligonucleotide chemistries that enhanceoligonucleotide:[target nucleic acid] duplexes are known in the art (seefor example, Freier et al., Nucleic Acids Research, 1997, 25, 4429). Aswill be appreciated by those skilled in the art, differentoligonucleotide chemistries may be preferred for different targetnucleic acids. That is, the optimal oligonucleotide chemistry forbinding to a target DNA might be suboptimal for binding to a target RNAhaving the same nucleotide sequence.

[0073] In effecting the process of the invention in the order step 300before step 400 as seen in FIG. 1, from a target nucleic acid sequenceassembled at step 200, a list of oligonucleotide sequences is generatedas represented in the flowchart shown in FIGS. 4 and 5. In step 302, thedesired oligonucleotide length is chosen. In a preferred embodiment,oligonucleotide length is between from about 8 to about 30, morepreferably from about 12 to about 25, nucleotides. In step 304, allpossible oligonucleotide sequences of the desired length capable ofhybridizing to the target sequence obtained in step 200 are generated.In this step, a series of oligonucleotide sequences are generated,simply by determining the most 5′ oligonucleotide possible and ‘walking’the target sequence in increments of one base until the 3′ mostoligonucleotide possible is reached.

[0074] In step 305, a virtual oligonucleotide chemistry is applied tothe nucleobase sequences of step 304 in order to yield a set of virtualoligonucleotides that can be evaluated in silico. Default virtualoligonucleotide chemistries include those that are well-characterized interms of their physical and chemical properties, e.g.,2′-deoxyribonucleic acid having naturally occurring bases (A, T, C andG), unmodified sugar residues and a phosphodiester backbone.

[0075] 4. In Silico Evaluation of Thermodynamic Properties of VirtualOligonucleotides.

[0076] In step 306, a series of thermodynamic, sequence, and homologyscores are preferably calculated for each virtual oligonucleotideobtained from step 305. Thermodynamic properties are calculated asrepresented in FIG. 6. In step 308, the desired thermodynamic propertiesare selected.

[0077] This will typically include step 309, calculation of the freeenergy of the target structure. If the oligonucleotide is a DNAmolecule, then steps 310, 312, and 314 are performed. If theoligonucleotide is an RNA molecule, then steps 311, 313 and 315 areperformed. In both cases, these steps correspond to calculation of thefree energy of intramolecular oligonucleotide interactions,intermolecular interactions and duplex formation. In addition, a freeenergy of oligonucleotide-target binding is preferably calculated atstep 316.

[0078] Other thermodynamic and kinetic properties may be calculated foroligonucleotides as represented at step 317.

[0079] Such other thermodynamic and kinetic properties may includemelting temperatures, association rates, dissociation rates, or anyother physical property that may be predictive of oligonucleotideactivity.

[0080] The free energy of the target structure is defined as the freeenergy needed to disrupt any secondary structure in the target bindingsite of the targeted nucleic acid. This region includes any intra-targetnucleotide base pairs that need to be disrupted in order for anoligonucleotide to bind to its complementary sequence. The effect ofthis localized disruption of secondary structure is to provideaccessibility by the oligonucleotide. Such structures will includedouble helices, terminal unpaired and mismatched nucleotides, loops,including hairpin loops, bulge loops, internal loops and multibranchloops (Serra et al., Methods in Enzymology, 1995, 259, 242).

[0081] The intermolecular free energies refer to inherent energy due tothe most stable structure formed by two oligonucleotides; suchstructures include dimer formation. Intermolecular free energies shouldalso be taken into account when, for example, two or moreoligonucleotides, of different sequence are to be administered to thesame cell in an assay.

[0082] The intramolecular free energies refer to the energy needed todisrupt the most stable secondary structure within a singleoligonucleotide. Such structures include, for example, hairpin loops,bulges and internal loops. The degree of intramolecular base pairing isindicative of the energy needed to disrupt such base pairing.

[0083] The free energy of duplex formation is the free energy ofdenatured oligonucleotide binding to its denatured target sequence. Theoligonucleotide-target binding is the total binding involved, andincludes the energies involved in opening up intra- and inter-molecularoligonucleotide structures, opening up target structure, and duplexformation.

[0084] The most stable RNA structure is predicted based on nearestneighbor analysis (Serra et al., Methods in Enzymology, 1995, 259, 242).This analysis is based on the assumption that stability of a given basepair is determined by the adjacent base pairs. For each possible nearestneighbor combination, thermodynamic properties have been determined andare provided. For double helical regions, two additional factors need tobe considered, an entropy change required to initiate a helix and aentropy change associated with self-complementary strands only. Thus,the free energy of a duplex can be calculated using the equation:

ΔG° _(T) =ΔH°−TΔS°

[0085] where:

[0086] ΔG is the free energy of duplex formation,

[0087] ΔH is the enthalpy change for each nearest neighbor,

[0088] ΔS is the entropy change for each nearest neighbor, and

[0089] T is temperature.

[0090] The ΔH and ΔS for each possible nearest neighbor combination havebeen experimentally determined. These letter values are often availablein published tables. For terminal unpaired and mismatched nucleotides,enthalpy and entropy measurements for each possible nucleotidecombination are also available in published tables. Such results areadded directly to values determined for duplex formation. For loops,while the available data is not as complete or accurate as for basepairing, one known model determines the free energy of loop formation asthe sum of free energy based on loop size, the closing base pair, theinteractions between the first mismatch of the loop with the closingbase pair, and additional factors including being closed by AU or UA ora first mismatch of GA or UU. Such equations may also be used foroligoribonucleotide-target RNA interactions.

[0091] The stability of DNA duplexes is used in the case of intra- orintermolecular oligodeoxyribonucleotide interactions. DNA duplexstability is calculated using similar equations as RNA stability, exceptexperimentally determined values differ between nearest neighbors in DNAand RNA and helix initiation tends to be more favorable in DNA than inRNA (SantaLucia et al., Biochemistry, 1996, 35, 3555).

[0092] Additional thermodynamic parameters are used in the case ofRNA/DNA hybrid duplexes. This would be the case for an RNA target andoligodeoxynucleotide. Such parameters were determined by Sugimoto et al.(Biochemistry, 1995, 34, 11211). In addition to values for nearestneighbors, differences were seen for values for enthalpy of helixinitiation.

[0093] 5. In Silico Evaluation of Target Accessibility

[0094] Target accessibility is believed to be an important considerationin selecting oligonucleotides. Such a target site will possess minimalsecondary structure and thus, will require minimal energy to disruptsuch structure. In addition, secondary structure in oligonucleotides,whether inter- or intra-molecular, is undesirable due to the energyrequired to disrupt such structures. Oligonucleotide-target binding isdependent on both these factors. It is desirable to minimize thecontributions of secondary structure based on these factors. The othercontribution to oligonucleotide-target binding is binding affinity.Favorable binding affinities based on tighter base pairing at the targetsite is desirable.

[0095] Following the calculation of thermodynamic properties ending atstep 317, the desired sequence properties to be scored are selected atstep 324. These properties include the number of strings of fourguanosine residues in a row at step 325) or three guanosines in a row atstep 326), the length of the longest string of adenosines at step 327),cytidines at step 328) or uridines or thymidines at step 329), thelength of the longest string of purines at step 330) or pyrimidines atstep 331), the percent composition of adenosine at step 332), cytidineat step 333), guanosine at step 334) or uridines or thymidines at step335, the percent composition of purines at step 336) or pyrimidines atstep 337), the number of CG dinucleotide repeats at step 338), CAdinucleotide repeats at step 339) or UA or TA dinucleotide repeats atstep 340). In addition, other sequence properties may be used as foundto be relevant and predictive of antisense efficacy, as represented atstep 341.

[0096] These sequence properties may be important in predictingoligonucleotide activity, or lack thereof. For example, U.S. Pat. No.5,523,389 discloses oligonucleotides containing stretches of three orfour guanosine residues in a row. Oligonucleotides having such sequencesmay act in a sequence-independent manner. For an antisense approach,such a mechanism is not usually desired. In addition, high numbers ofdinucleotide repeats may be indicative of low complexity regions whichmay be present in large numbers of unrelated genes. Unequal basecomposition, for example, 90% adenosine, can also give non-specificeffects. From a practical standpoint, it may be desirable to removeoligonucleotides that possess long stretches of other nucleotides due tosynthesis considerations. Other sequences properties, either listedabove or later found to be of predictive value may be used to selectoligonucleotide sequences.

[0097] Following step 341, the homology scores to be calculated areselected in step 342. Homology to nucleic acids encoding proteinisoforms of the target, as represented at step 343, may be desired. Forexample, oligonucleotides specific for an isoform of protein kinase Ccan be selected. Also, oligonucleotides can be selected to targetmultiple isoforms of such genes. Homology to analogous target sequences,as represented at step 344, may also be desired. For example, anoligonucleotide can be selected to a region common to both humans andmice to facilitate testing of the oligonucleotide in both species.Homology to splice variants of the target nucleic acid, as representedat step 345, may be desired. In addition, it may be desirable todetermine homology to other sequence variants as necessary, asrepresented in step 346.

[0098] Following step 346, from which scores were obtained in eachselected parameter, a desired range is selected to select the mostpromising oligonucleotides, as represented at step 347. Typically, onlyseveral parameters will be used to select oligonucleotide sequences. Asstructure prediction improves, additional parameters may be used. Oncethe desired score ranges are chosen, a list of all oligonucleotideshaving parameters falling within those ranges will be generated, asrepresented at step 348.

[0099] 6. Targeting Oligonucleotides to Functional Regions of a NucleicAcid.

[0100] It may be desirable to target oligonucleotide sequences tospecific functional regions of the target nucleic acid. A decision ismade whether to target such regions, as represented in decision step349. If it is desired to target functional regions then process step 350occurs as seen in greater detail in FIG. 9. If it is not desired thenthe process proceeds to step 375.

[0101] In step 350, as seen in FIG. 9, the desired functional regionsare selected. Such regions include the transcription start site or 5′cap at step 353), the 5′ untranslated region at step 354), the startcodon at step 355, the coding region at step 356), the stop codon atstep 357), the 3′ untranslated region at step 358), 5′ splice sites atstep 359 or 3′ splice sites at step 360, specific exons at step 361) orspecific introns at step 362), mRNA stabilization signal at step 363),mRNA destabilization signal at step 364), poly-adenylation signal atstep 365), poly-A addition site at step 366), poly-A tail at step 367),or the gene sequence 5′ of known pre-mRNA at step 368). In addition,additional functional sites may be selected, as represented at step 369.

[0102] Many functional regions are important to the proper processing ofthe gene and are attractive targets for antisense approaches. Forexample, the AUG start codon is commonly targeted because it isnecessary to initiate translation. In addition, splice sites are thoughtto be attractive targets because these regions are important forprocessing of the mRNA. Other known sites may be more accessible becauseof interactions with protein factors or other regulatory molecules.

[0103] After the desired functional regions are selected and determined,then a subset of all previously selected oligonucleotides are selectedbased on hybridization to only those desired functional regions, asrepresented by step 370.

[0104] 7. Uniform Distribution of Oligonucleotides.

[0105] Whether or not targeting functional sites is desired, a largenumber of oligonucleotide sequences may result from the process thusfar. In order to reduce the number of oligonucleotide sequences to amanageable number, a decision is made whether to uniformly distributeselected oligonucleotides along the target, as represented in step 375.A uniform distribution of oligonucleotide sequences will aim to providecomplete coverage throughout the complete target nucleic acid or theselected functional regions. A utility is used to automate thedistribution of sequences, as represented in step 380. Such a utilityfactors in parameters such as length of the target nucleic acid, totalnumber of oligonucleotide sequences desired, oligonucleotide sequencesper unit length, number of oligonucleotide sequences per functionalregion. Manual selection of oligonucleotide sequences is also providedfor by step 385. In some cases, it may be desirable to manually selectoligonucleotide sequences. For example, it may be useful to determinethe effect of small base shifts on activity. Once the desired number ofoligonucleotide sequences is obtained either from step 380 or step 385,then these oligonucleotide sequences are passed onto step 400 of theprocess, where oligonucleotide chemistries are assigned.

[0106] 8. Assignment of Actual Oligonucleotide Chemistry.

[0107] Once a set of select nucleobase sequences has been generatedaccording to the preceding process and decision steps, actualoligonucleotide chemistry is assigned to the sequences. An ‘actualoligonucleotide chemistry’ or simply ‘chemistry’ is a chemical motifthat is common to a particular set of robotically synthesizedoligonucleotide compounds. Preferred chemistries include, but are notlimited to, oligonucleotides in which every linkage is aphosphorothioate linkage, and chimeric oligonucleotides in which adefined number of 5′ and/or 3′ terminal residues have a 2′-methoxyethoxymodification.

[0108] Chemistries can be assigned to the nucleobase sequences duringgeneral procedure step 400 (FIG. 1). The logical basis for chemistryassignment is illustrated in FIGS. 10 and 11 and an iterative routinefor stepping through an oligonucleotide nucleoside by nucleoside isillustrated in FIG. 12. Chemistry assignment can be effected byassignment directly into a word processing program, via an interactiveword processing program or via automated programs and devices. In eachof these instances, the output file is selected to be in a format thatcan serve as an input file to automated synthesis devices.

[0109] 9. Oligonucleotide Compounds.

[0110] In the context of this invention, in reference tooligonucleotides, the term ‘oligonucleotide’ is used to refer to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics thereof. Thus this term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native forms,i.e., phosphodiester linked A, C, G, T and U nucleosides, because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target and increased stability in thepresence of nucleases.

[0111] The oligonucleotide compounds in accordance with this inventioncan be of various lengths depending on various parameters, including butnot limited to those discussed above in reference to the selectioncriteria of general procedure 300. For use as antisense oligonucleotidescompounds of the invention preferably are from about 8 to about 30nucleobases in length. Particularly preferred are antisenseoligonucleotides comprising from about 12 to about 25 nucleobases (i.e.from about 8 to about 30 linked nucleosides). A discussion of antisenseoligonucleotides and some desirable modifications can be found in DeMesmaeker et al., Acc. Chem. Res., 1995, 28, 366. Other lengths ofoligonucleotides might be selected for non-antisense targetingstrategies, for instance using the oligonucleotides as ribozymes. Suchribozymes normally require oligonucleotides of longer length as is knownin the art.

[0112] A nucleoside is a base-sugar combination. The base portion of thenucleoside is normally a heterocyclic base. The two most common classesof such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a normal (where normal is defined as beingfound in RNA and DNA) pentofuranosyl sugar, the phosphate group can belinked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure, however, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal linkage or backbone of RNA and DNA isa 3′ to 5′ phosphodiester linkage.

[0113] Specific examples of preferred oligonucleotides useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0114] 10. Selection of Oligonucleotide Chemistries.

[0115] In a general logic scheme as illustrated in FIGS. 10 and 11, foreach nucleoside position, the user or automated device is interrogatedfirst for a base assignment, followed by a sugar assignment, a linkerassignment and finally a conjugate assignment. Thus for each nucleoside,at process step 410 a base is selected. In selecting the base, basechemistry 1 can be selected at process step 412 or one or morealternative bases are selected at process steps 414, 416 and 418. Afterbase selection is effected, the sugar portion of the nucleoside isselected. Thus for each nucleoside, at process step 420 a sugar isselected that together with the select base will complete thenucleoside. In selecting the sugar, sugar chemistry 1 can be selected atprocess 422 or one or more alternative sugars are selected at processsteps 424, 426 and 428. For each two adjacent nucleoside units, atprocess step 430, the internucleoside linker is selected. The linkerchemistry for the internucleoside linker can be linker chemistry 1selected at process step 432 or one or more alternative internucleosidelinker chemistries are selected at process steps 434, 436 and 438.

[0116] In addition to the base, sugar and internucleoside linkage, ateach nucleoside position, one or more conjugate groups can be attachedto the oligonucleotide via attachment to the nucleoside or attachment tothe internucleoside linkage. The addition of a conjugate group isintegrated at process step 440 and the assignment of the conjugate groupis effected at process step 450.

[0117] For illustrative purposes in FIGS. 10 and 11, for each of thebase, the sugar, the internucleoside linkers, or the conjugate,chemistries 1 though n are illustrated. As described in thisspecification, it is understood that the number of alternate chemistriesbetween chemistry 1 and alternative chemistry n, for each of the base,the sugar, the internucleoside linkage and the conjugate, is variableand includes, but is not limited to, each of the specific alternativebases, sugar, internucleoside linkers and conjugates identified in thisspecification as well as equivalents known in the art.

[0118] Utilizing the logic as described in conjunction with FIGS. 10 and11, chemistry is assigned, as is shown in FIG. 12, to the list ofoligonucleotides from general procedure 300. In assigning chemistries tothe oligonucleotides in this list, a pointer can be set at process step452 to the first oligonucleotide in the list and at step 453 to thefirst nucleotide of that first oligonucleotide. The base chemistry isselected at step 410, as described above, the sugar chemistry isselected at step 420, also as described above, followed by selection ofthe internucleoside linkage at step 430, also as described above. Atdecision 440, the process branches depending on whether a conjugate willbe added at the current nucleotide position. If a conjugate is desired,the conjugate is selected at step 450, also as described above.

[0119] Whether or not a conjugate was added at decision step 440, aninquiry is made at decision step 454. This inquiry asks if the pointerresides at the last nucleotide in the current oligonucleotide. If theresult at decision step 454 is ‘No’, the pointer is moved to the nextnucleotide in the current oligonucleotide and the loop including steps410, 420, 430, 440 and 454 is repeated. This loop is reiterated untilthe result at decision step 454 is ‘Yes.’

[0120] When the result at decision step 454 is ‘Yes’, a query is made atdecision step 460 concerning the location of the pointer in the list ofoligonucleotides. If the pointer is not at the last oligonucleotide ofthe list, the ‘No’ path of the decision step 460 is followed and thepointer is moved to the next oligonucleotide in the list at process step458. With the pointer set to the next oligonucleotide in the list, theloop that starts at process steps 453 is reiterated. When the result atdecision step 460 is ‘Yes’, chemistry has been assigned to all of thenucleotides in the list of oligonucleotides.

[0121] 11. Description of Oligonucleotide Chemistries.

[0122] As is illustrated in FIG. 10, for each nucleoside of anoligonucleotide, chemistry selection includes selection of the baseforming the nucleoside from a large palette of different base unitsavailable. These may be ‘modified’ or ‘natural’ bases (also referenceherein as nucleobases) including the natural purine bases adenine (A)and guanine (G), and the natural pyrimidine bases thymine (T), cytosine(C) and uracil (U). They further can include modified nucleobasesincluding other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-halo uracilsand cytosines particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred for selection as the base. These are particularlyuseful when combined with a 2¹-methoxyethyl sugar modifications,described below.

[0123] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, Reference is also made to allowedU.S. patent application Ser. No. 08/762,488, filed on Dec. 10, 1996,commonly owned with the present application and herein incorporated byreference.

[0124] In selecting the base for any particular nucleoside of anoligonucleotide, consideration is first given to the need of a base fora particular specificity for hybridization to an opposing strand of aparticular target. Thus if an ‘A’ base is required, adenine might beselected however other alternative bases that can effect hybridizationin a manner mimicking an ‘A’ base such as 2,6-diaminopurine might beselected should other consideration, e.g., stronger hybridization(relative to hybridization achieved with adenine), be desired.

[0125] As is illustrated in FIG. 10, for each nucleoside of anoligonucleotide, chemistry selection includes selection of the sugarforming the nucleoside from a large palette of different sugar or sugarsurrogate units available. These may be modified sugar groups, forinstance sugars containing one or more substituent groups. Preferredsubstituent groups comprise the following at the 2′ position: OH; F; O—,S—, or N-alkyl, O—, S—, or N-alkenyl, or O, S— or N-alkynyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n), CH₃,O(CH₂)_(n)ONH_(2,) and O (CH₂)_(n)ON [(CH₂)_(n)CH₃)]₂, where n and m arefrom 1 to about 10. Other preferred substituent groups comprise one ofthe following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂₀CH₃,also known as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylamino oxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in co-ownedU.S. patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, thecontents of which are herein incorporated by reference.

[0126] Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the sugar group,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide. The nucleosides of the oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

[0127] Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the present application, each of which is herein incorporated byreference, together with allowed U.S. patent application Ser. No.08/468,037, filed on Jun. 5, 1995, which is commonly owned with thepresent application and is herein incorporated by reference.

[0128] As is illustrated in FIG. 10, for each adjacent pair ofnucleosides of an oligonucleotide, chemistry selection includesselection of the internucleoside linkage. These internucleoside linkagesare also referred to as linkers, backbones or oligonucleotide backbones.For forming these nucleoside linkages, a palette of differentinternucleoside linkages or backbones is available. These includemodified oligonucleotide backbones, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotri-esters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalklyphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

[0129] Representative United States patents that teach the preparationof the above phosphorus containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

[0130] Preferred internucleoside linkages for oligonucleotides that donot include a phosphorus atom therein, i.e., for oligonucleosides, havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, 0, S and CH₂ component parts.

[0131] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0132] In other preferred oligonucleotides, i.e., oligo-nucleotidemimetics, both the sugar and the intersugar linkage, i.e., the backbone,of the nucleotide units are replaced with novel groups. The base unitsare maintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an oligonucleotide mimetic thathas been shown to have excellent hybridization properties, is referredto as a peptide nucleic acid (PNA). In PNA compounds, thesugar-phosphate backbone of an oligonucleotide is replaced with anamide-containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S.: 5,539,082; 5,714,331; and5,719,262, each of which is herein incorporated by reference. Furtherteaching of PNA compounds can be found in Nielsen et al., Science, 1991,254, 1497.

[0133] For the internucleoside linkages, the most preferred embodimentsof the invention are oligonucleotides with phosphorothioate backbonesand oligonucleosides with heteroatom backbones, and in particular—CH₂—NH—O—CH₂—, —CH₂—N(CH3)—O—CH₂— [known as a methylene (methylimino)or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0134] In attaching a conjugate group to one or more nucleosides orinternucleoside linkages of an oligo-nucleotide, various properties ofthe oligonucleotide are modified. Thus modification of theoligonucleotides of the invention to chemically link one or moremoieties or conjugates to the oligonucleotide are intended to enhancethe activity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Let., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci.; 1992, 660, 306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327;Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0135] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the present application, andeach of which is herein incorporated by reference.

[0136] 12. Chimeric Compounds.

[0137] It is not necessary for all positions in a given compound to beuniformly modified. In fact, more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes compounds which are chimeric compounds. ‘Chimeric’ compounds or‘chimeras,’ in the context of this invention, are compounds,particularly oligonucleotides, which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.

[0138] By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0139] Chimeric antisense compounds of the invention may be formed ascomposite structures representing the union of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide mimetics as described above. Such compounds have alsobeen referred to in the art as “hybrids” or “gapmers”. RepresentativeUnited States patents that teach the preparation of such hybridstructures include, but are not limited to, U.S. Pat. Nos. 5,013,830;5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain ofwhich are commonly owned with the present application and each of whichis herein incorporated by reference, together with commonly owned andallowed U.S. patent application Ser. No. 08/465,880, filed on Jun. 6,1995, also herein incorporated by reference.

[0140] 13. Description of Automated Oligonucleotide Synthesis.

[0141] In the next step of the overall process (illustrated in FIGS. 1and 2), oligonucleotides are synthesized on an automated synthesizer.Although many devices may be employed, the synthesizer is preferably avariation of the synthesizer described in U.S. Pat. Nos. 5,472,672 and5,529,756, the entire contents of which are herein incorporated byreference. The synthesizer described in those patents is modified toinclude movement in along the Y axis in addition to movement along the Xaxis. As so modified, a 96-well array of compounds can be synthesized bythe synthesizer. The synthesizer further includes temperature controland the ability to maintain an inert atmosphere during all phases ofsynthesis. The reagent array delivery format employs orthogonal X-axismotion of a matrix of reaction vessels and Y-axis motion of an array ofreagents. Each reagent has its own dedicated plumbing system toeliminate the possibility of cross-contamination of reagents and lineflushing and/or pipette washing. This in combined with a high deliveryspeed obtained with a reagent mapping system allows for the extremelyrapid delivery of reagents. This further allows long and complexreaction sequences to be performed in an efficient and facile manner.

[0142] The software that operates the synthesizer allows thestraightforward programming of the parallel synthesis of a large numberof compounds. The software utilizes a general synthetic procedure in theform of a command (.cmd) file, which calls upon certain reagents to beadded to certain wells via lookup in a sequence (.seq) file. The bottleposition, flow rate, and concentration of each reagent is stored in alookup table (.tab) file. Thus, once any synthetic method has beenoutlined, a plate of compounds is made by permutating a set of reagents,and writing the resulting output to a text file. The text file is inputdirectly into the synthesizer and used for the synthesis of the plate ofcompounds. The synthesizer is interfaced with a relational databaseallowing data output related to the synthesized compounds to beregistered in a highly efficient manner.

[0143] Building of the seq, .cmd and tab files is illustrated in FIG.13. Thus as a part of the general oligonucleotide synthesis procedure500, for each linker chemistry at process step 502, a synthesis file,i.e., a cmd file, is built at process step 504. This file can be builtfresh to reflect a completely new set of machine commands reflecting aset of chemical synthesis steps or it can modify an existing file storedat process step 504 by editing that stored file in process step 508. The.cmd files are built using a word processor and a command set ofinstructions as outlined below.

[0144] It will be appreciated that the preparation of control softwareand data files is within the routine skill of persons skilled inanotated nucleotide synthesis. The same will depend upon the hardwareemployed, the chemistries adopeted and the design paradigm selected bythe operator.

[0145] In a like manner to the building the .cmd files, tab files arebuilt to reflect the necessary reagents used in the automaticsynthesizer for the particular chemistries that have been selected forthe linkages, bases, sugars and conjugate chemistries. Thus for each ofa set of these chemistries at process step 510, a tab file is built atprocess step 512 and stored at process step 514. As with the .cmd files,an existing tab file can be edited at process step 516.

[0146] Both the .cmd files and the tab files are linked together atprocess step 518 and stored for later retrieval in an appropriate sampledatabase 520. Linking can be as simple as using like file names toassociate a .cmd file to its appropriate tab file, e.g., synthesis_l.cmdis linked to synthesis l.tab by use of the same preamble in their names.

[0147] The automated, multi-well parallel array synthesizer employs areagent array delivery format, in which each reagent utilized has adedicated plumbing system. As seen in FIGS. 23 and 24, an inertatmosphere 522 is maintained during all phases of a synthesis.Temperature is controlled via a thermal transfer plate 524, which holdsan injection molded reaction block 526. The reaction plate assemblyslides in the X-axis direction, while for example eight nozzle blocks(528, 530, 532, 534, 536, 538, 540 and 542) holding the reagent linesslide in the Y-axis direction, allowing for the extremely rapid deliveryof any of 64 reagents to 96 wells. In addition, there are for example,six banks of fixed nozzle blocks (544, 546, 548, 550, 552 and 554) whichdeliver the same reagent or solvent to eight wells at once, for a totalof 72 possible reagents.

[0148] In synthesizing oligonucleotides for screening, the targetreaction vessels, a 96 well plate 556 (a 2-dimensional array), moves inone direction along the X axis, while the series of independentlycontrolled reagent delivery nozzles (528, 530, 532, 534, 536, 538, 540and 542) move along the Y-axis relative to the reaction vessel 558. Asthe reaction plate 556 and reagent nozzles (528, 530, 532, 534, 536,538, 540 and 542) can be moved independently at the same time, thisarrangement facilitated the extremely rapid delivery of up to 72reagents independently to each of the 96 reaction vessel wells.

[0149] The system software allows the straightforward programming of thesynthesis of a large number of compounds by supplying the generalsynthetic procedure in the form of the command file to call upon certainreagents to be added to specific wells via lookup in the sequence filewith the bottle position, flow rate, and concentration of each reagentbeing stored in the separate reagent table file. Compounds can besynthesized on various scales. For oligonucleotides, a 200 nmole scaleis typically selected while for other compounds larger scales, as forexample a 10 μmole scale (3-5 mg), might be utilized. The resultingcrude compounds are generally >80% pure, and are utilized directly forhigh throughput screening assays. Alternatively, prior to use the platescan be subjected to quality control (see general procedure 600 andExample 9) to ascertain their exact purity. Use of the synthesizerresults in a very efficient means for the parallel synthesis ofcompounds for screening.

[0150] The software inputs accept tab delimited text files (as discussedabove for file 504 and 512) from any text editor. A typical commandfile, a .cmd file, is shown in Example 3 at Table 2. Typical sequencefiles, seq files, are shown in Example 3 at Tables 3 and 4 (.SEQ file),and a typical reagent file, a tab file, is shown in Example 3 at Table5. Table 3 illustrates the sequence file for an oligonucleotide having2′-deoxy nucleotides at each position with a phosphorothioate backbonethroughout. Table 4 illustrates the sequence file for anoligonucleotide, again having a phosphorothioate backbone throughout,however, certain modified nucleoside are utilized in portions of theoligonucleotide. As shown in this table, 2′—O—(methoxyethyl) modifiednucleoside are utilized in a first region (a wing) of theoligonucleotide, followed by a second region (a gap) of 2′-deoxynucleotides and finally a third region (a further wing) that has thesame chemistry as the first region. Typically some of the wells of the96 well plate 556 may be left empty (depending on the number ofoligonucleotides to be made during an individual synthesis) or some ofthe well may have oligonucleotides that will serve as standards forcomparison or analytical purposes.

[0151] Prior to loading reagents, moisture sensitive reagent lines arepurged with argon at 522 for 20 minutes. Reagents are dissolved toappropriate concentrations and installed on the synthesizer. Largebottles, collectively identified as 0.558 in FIG. 23 (containing 8delivery lines) are used for wash solvents and the delivery of generalactivators, trityl group cleaving reagents and other reagents that maybe used in multiple wells during any particular synthesis. Small septabottles, collectively identified as 560 in FIG. 23, are utilized tocontain individual nucleotide amidite precursor compounds. This allowsfor anhydrous preparation and efficient installation of multiplereagents by using needles to pressurize the bottle, and as a deliverypath. After all reagents are installed, the lines are primed withreagent, flow rates measured, then entered into the reagent table (.tabfile). A dry resin loaded plate is removed from vacuum and installed inthe machine for the synthesis.

[0152] The modified 96 well polypropylene plate 556 is utilized as thereaction vessel. The working volume in each well is approximately 700μl. The bottom of each well is provided with a pressed-fit 20 μmpolypropylene frit and a long capillary exit into a lower collectionchamber as is illustrated in FIG. 5 of the above referenced U.S. Pat.No. 5,372,672. The solid support for use in holding the growingoligonucleotide during synthesis is loaded into the wells of thesynthesis plate 556 by pipetting the desired volume of a balanceddensity slurry of the support suspended in an appropriate solvent,typically an acetonitrile-methylene chloride mixture. Reactions can berun on various scales as for instance the above noted 200 nmole and 10μmol scales. For oligonucleotide synthesis a CPG support is preferred,however other medium loading polystyrene-PEG supports such as TentaGel™or ArgoGel™ can also be used.

[0153] As seen in FIG. 24, the synthesis plate is transported back andforth in the X-direction under an array of 8 moveable banks (530, 532,534, 536, 538, 540, 542 and 544) of 8 nozzles (64 total) in theY-direction, and 6 banks (544, 546, 548, 550, 552 and 554) of 48 fixednozzles, so that each well can receive the appropriate amounts ofreagents and/or solvents from any reservoir (large bottle or smallersepta bottle). A sliding balloon-type seal 562 surrounds this nozzlearray and joins it to the reaction plate headspace 564. A slow sweep ofnitrogen or argon 522 at ambient pressure across the plate headspace isused to preserve an anhydrous environment.

[0154] The liquid contents in each well do not drip out until theheadspace pressure exceeds the capillary forces on the liquid in theexit nozzle. A slight positive pressure in the lower collection chambercan be added to eliminate residual slow leakage from filled wells, or toeffect agitation by bubbling inert gas through the suspension. In orderto empty the wells, the headspace gas outlet valve is closed and theinternal pressure raised to about 2 psi.

[0155] Normally, liquid contents are blown directly to waste 566.

[0156] However, a 96 well microtiter plate can be inserted into thelower chamber beneath the synthesis plate in order to collect theindividual well eluents for spectrophotometric monitoring (trityl, etc.)of reaction progress and yield.

[0157] The basic plumbing scheme for the machine is the gas-pressurizeddelivery of reagents. Each reagent is delivered to the synthesis platethrough a dedicated supply line, collectively identified at 568,solenoid valve collectively identified at 570 and nozzle, collectivelyidentified at 572. Reagents never cross paths until they reach thereaction well. Thus, no line needs to be washed or flushed prior to itsnext use and there is no possibility of cross-contamination of reagents.The liquid delivery velocity is sufficiently energetic to thoroughly mixthe contents within a well to form a homogeneous solution, even whenemploying solutions having drastically different densities. With thismixing, once reactants are in homogeneous solution, diffusion carriesthe individual components into and out of the solid support matrix wherethe desired reaction takes place. Each reagent reservoir can be plumbedto either a single nozzle or any combination of up to 8 nozzles. Eachnozzle is also provided with a concentric nozzle washer to wash theoutside of the delivery nozzles in order to eliminate problems ofcrystallized reactant buildup due to slow evaporation of solvent at thetips of the nozzles. The nozzles and supply lines can be primed into aset of dummy wells directly to waste at any time.

[0158] The entire plumbing system is fabricated with teflon tubing, andreagent reservoirs are accessed via syringe needle/septa or directconnection into the higher capacity bottles. The septum vials 560 areheld in removable 8-bottle racks to facilitate easy setup and cleaning.The priming volume for each line is about 350 μl. The minimum deliveryvolume is about 2 μl, and flow rate accuracy is ±5%. The actual amountof material delivered depends on a timed flow of liquid. The flow ratefor a particular solvent will depend on its viscosity and wettingcharacteristics of the teflon tubing. The flow rate (typically 200-350μl per sec) is experimentally determined, and this information iscontained in the reagent table setup file.

[0159] Heating and cooling of the reaction block 526 is effectedutilizing a recirculating heat exchanger plate 524, similar to thatfound in PCR thermocyclers, that nests with the polypropylene synthesisplate 556 to provide good thermal contact. The liquid contents in a wellcan be heated or cooled at about 10° C. per minute over a range of +5 to+80° C., as polypropylene begins to soften and deform at about 80° C.For temperatures greater than this, a non-disposable synthesis platemachined from stainless steel or monel with replaceable frits can beutilized.

[0160] The hardware controller can be any of a wide variety, butconveniently can be designed around a set of three 1 MHz 86332 chips.This controller is used to drive the single X-axis and 8 Y-axis steppermotors as well as provide the timing functions for a total of 154solenoid valves. Each chip has 16 bidirectional timer I/O and 8interrupt channels in its timer processing unit (TPU). These are used toprovide the step and direction signals, and to read 3 encoder inputs and2 limit switches for controlling up to three motors per chip. Each 86332chip also drives a serial chain of 8 UNC5891A darlington array chips toprovide power to 64 valves with msec resolution. The controllercommunicates with the Windows software interface program running on a PCvia a 19200 Hz serial channel, and uses an elementary instruction set tocommunicate valve_number, time_open, motor_number and position_data.

[0161] The three components of the software program that run the arraysynthesizer, the generalized procedure or command (.cmd) file whichspecifies the synthesis instructions to be performed, the sequence(.seq) file which specifies the scale of the reaction and the order inwhich variable groups will be added to the core synthon, and the reagenttable (.tab) file which specifies the name of a chemical, its location(bottle number), flow rate, and concentration are utilized inconjunction with a basic set of command instructions.

[0162] One basic set of command instructions can be: ADD IF {block ofinstructions} END_IF REPEAT {block of instructions} END_REPEAT PRIME,NOZZLE_WASH WAIT, DRAIN LOAD, REMOVE NEXT_SEQUENCE LOOP_BEGIN, LOOP_END

[0163] The ADD instruction has two forms, and is intended to have thelook and feel of a standard chemical equation. Reagents are specified tobe added by a molar amount if the number proceeds the name identifier,or by an absolute volume in microliters if the number follows theidentifier. The number of reagents to be added is a parsed list,separated by the ‘+’ sign. For variable reagent identifiers, the keyword, <seq>, means look in the sequence table for the identity of thereagent to be added, while the key word, <act>, means add the reagentwhich is associated with that particular <seq>. Reagents are deliveredin the order specified in the list.

[0164] Thus:

[0165] ADD ACN 300

[0166] means: Add 300 μl of the named reagent acetonitrile; ACN to eachwell of active synthesis

[0167] ADD <seq>300

[0168] means: If the sequence pointer in the seq file is to a reagent inthe list of reagents, independent of scale, add 300 μl of thatparticular reagent specified for that well.

[0169] ADD 1.1 PYR +1.0 <seq>+1.1 <actl>

[0170] means: If the sequence pointer in the seq file is to a reagent inthe list of acids in the Class ACIDS_(—)1, and PYR is the name ofpyridine, and ethyl chloroformate is defined in the tab file to activatethe class, ACIDS_(—)1, then this instruction means:

[0171] Add 1.1 equiv. pyridine

[0172] 1.0 equiv. of the acid specified for that well and

[0173] 1.1 equiv. of the activator, ethyl chloroformate

[0174] The IF command allows one to test what type of reagent isspecified in the <seq>variable and process the succeeding block ofcommands accordingly. Thus: ACYLATION   {the procedure name} BEGIN IFCLASS = ACIDS_1 ADD 1.0 <seq> + 1.1 <act1> + 1.1 PYR WAIT 60 ENDIF IFCLASS = ACIDS_2 ADD 1.0 <seq> + 1.2 <act1> + 1.2 TEA ENDIF WAIT 60 DRAIN10 END

[0175] means: Operate on those wells for which reagents contained in theAcid_(—)1 class are specified, WAIT 60 sec, then operate on those wellsfor which reagents contained in the Acid_(—)2 class are specified, thenWAIT 60 sec longer, then DRAIN the whole plate. Note that the Acid_(—)1group has reacted for a total of 120 sec, while the Acid_(—)2 group hasreacted for only 60 sec.

[0176] The REPEAT command is a simple way to execute the same block ofcommands multiple times. Thus: WASH_1   {the procedure name} BEGINREPEAT 3 ADD ACN 300 DRAIN 15 END_REPEAT END

[0177] means: repeats the add acetonitrile and drain sequence for eachwell three times.

[0178] The PRIME command will operate either on specific named reagentsor on nozzles which will be used in the next associated <seq>operation.The μl amount dispensed into a prime port is a constant that can bespecified in a config.dat file.

[0179] The NOZZLE_WASH command for washing the outside of reactionnozzles free from residue due to evaporation of reagent solvent willoperate either on specific named reagents or on nozzles which have beenused in the preceding associated <seq>operation. The machine is plumbedsuch that if any nozzle in a block has been used, all the nozzles inthat block will be washed into the prime port.

[0180] The WAIT and DRAIN commands are by seconds, with the draincommand applying a gas pressure over the top surface of the plate inorder to drain the wells.

[0181] The LOAD and REMOVE commands are instructions for the machine topause for operator action.

[0182] The NEXT SEQUENCE command increments the sequence pointer to thenext group of substituents to be added in the sequence file. The generalform of a seq file entry is the definition:

[0183] Well_No Well_ID Scale Sequence

[0184] The sequence information is conveyed by a series of columns, eachof which represents a variable reagent to be added at a particularposition. The scale (μmole) variable is included so that reactions ofdifferent scale can be run at the same time if desired. The reagents aredefined in a lookup table (the .tab file), which specifies the name ofthe reagent as referred to in the sequence and command files, itslocation (bottle number), flow rate, and concentration. This informationis then used by the controller software and hardware to determine boththe appropriate slider motion to position the plate and slider arms fordelivery of a specific reagent, as well as the specific valve and timerequired to deliver the appropriate reagents. The adept classificationof reagents allows the use of conditional IF loops from within a commandfile to perform addition of different reagents differently during a‘single step’ performed across 96 wells simultaneously. The specialclass ACTIVATORS defines certain reagents that always get added with aparticular class of reagents (for example tetrazole during aphosphitylation reaction in adding the next nucleotide to a growingoligonucleotide).

[0185] The general form of the .tab file is the definition:

[0186] Class Bottle Reagent Name Flow_rate Conc.

[0187] The LOOP_BEGIN and LOOP_END commands define the block of commandswhich will continue to operate until a NEXT_SEQUENCE command points pastthe end of the longest list of reactants in any well.

[0188] Not included in the command set is a MOVE command. For all of theabove commands, if any plate or nozzle movement is required, this isautomatically executed in order to perform the desired solvent orreagent delivery operation. This is accomplished by the controllersoftware and hardware, which determines the correct nozzle(s) andwell(s) required for a particular reagent addition, then synchronizesthe position of the requisite nozzle and well prior to adding thereagent.

[0189] A MANUAL mode can also be utilized in which the synthesis plateand nozzle blocks can be ‘homed’ or moved to any position by theoperator, the nozzles primed or washed, the various reagent bottlesdepressurized or washed with solvent, the chamber pressurized, etc. Theautomatic COMMAND mode can be interrupted at any point, MANUAL commandsexecuted, and then operation resumed at the appropriate location. Thesequence pointer can be incremented to restart a synthesis anywherewithin a command file.

[0190] In reference to FIG. 14, the list of oligonucleotides forsynthesis can be rearranged or grouped for optimization of synthesis.Thus at process step 574, the oligonucleotides are grouped according toa factor on which to base the optimization of synthesis. As illustratedin the Examples below, one such factor is the 3′ most nucleoside of theoligonucleotide. Using the amidite approach for oligonucleotidesynthesis, a nucleotide bearing a 3′ phosphoramite is added to the 5′hydroxyl group of a growing nucleotide chain. The first nucleotide (atthe 3′ terminus of the oligonucleotide—the 3′ most nucleoside) is firstconnected to a solid support. This is normally done batchwise on a largescale as is standard practice during oligonucleotide synthesis.

[0191] Such solid supports pre-loaded with a nucleoside are commerciallyavailable. In utilizing the multi well format for oligonucleotidesynthesis, for each oligonucleotide to be synthesized, an aliquot of asolid support bearing the proper nucleoside thereon is added to the wellfor synthesis. Prior to loading the sequence of oligonucleotides to besynthesized in the seq file, they are sorted by the 3′ terminalnucleotide. Based on that sorting, all of the oligonucleotide sequenceshaving an ‘A’ nucleoside at their 3′ end are grouped together, thosewith a ‘C’ nucleoside are grouped together as are those with ‘G’ or ‘T’nucleosides. Thus in loading the nucleoside-bearing solid support intothe synthesis wells, machine movements are conserved.

[0192] The oligonucleotides can be grouped by the above describedparameter or other parameters that facilitate the synthesis of theoligonucleotides. Thus in FIG. 14, sorting is noted as being effected bysome parameter of type 1, as for instance the above described 3′ mostnucleoside, or other types of parameters from type 2 to type n atprocess steps 576, 578 and 580. Since synthesis will be from the 3′ endof the oligonucleotides to the 5′ end, the oligonucleotide sequences arereverse sorted to read 3′ to 5′. The oligonucleotides are entered in theseq file in this form, i.e., reading 3′ to 5′.

[0193] Once sorted into types, the position of the oligonucleotides onthe synthesis plates is specified at process step 582 by the creation ofa seq file as described above. The seq file is associated with therespective .cmd and tab files needed for synthesis of the particularchemistries specified for the oligonucleotides at process step 584 byretrieval of the .cmd and tab files at process step 586 from the sampledatabase 520. These files are then input into the multi well synthesizerat process step 588 for oligonucleotide synthesis. Once physicallysynthesized, the list of oligonucleotides again enters the generalprocedure flow as indicated in FIG. 1. For shipping, storage or otherhandling purposes, the plates can be lyophilized at this point ifdesired. Upon lyophilization, each well contains the oligonucleotideslocated therein as a dry compound.

[0194] 14. Quality Control.

[0195] In an optional step, quality control is performed on theoligonucleotides at process step 600 after a decision is made (decisionstep 550) to perform quality control. Although optional, quality controlmay be desired when there is some reason to doubt that some aspect ofthe synthetic process step 500 has been compromised. Alternatively,samples of the oligonucleotides may be taken and stored in the eventthat the results of assays conducted using the oligonucleotides (processstep 700) yield confusing results or suboptimal data. In the latterevent, for example, quality control might be performed after decisionstep 800 if no oligonucleotides with sufficient activity are identified.In either event, decision step 650 follows quality control step process600. If one or more of the oligonucleotides do not pass quality control,process step 500 can be repeated, i.e., the oligonucleotides aresynthesized for a second time.

[0196] The operation of the quality control system general procedure 600is detailed in steps 610-660 of FIG. 15. Also referenced in thefollowing discussion are the robotics and associated analyticalinstrumentation as shown in FIG. 18.

[0197] During step 610 (FIG. 15), sterile, double-distilled water istransferred by an automated liquid handler (2040 of FIG. 18) to eachwell of a multi-well plate containing a set of lyophilized antisenseoligonucleotides. The automated liquid handler (2040 of FIG. 18) readsthe barcode sticker on the multi-well plate to obtain the plate'sidentification number. Automated liquid handler 2040 then queries SampleDatabase 520 (which resides in Database Server 2002 of FIG. 18) for thequality control assay instruction set for that plate and executes theappropriate steps. Three quality control processes are illustrated,however, it is understood that other quality control processes or stepsmaybe practiced in addition to or in place of the processes illustrated.

[0198] The first illustrative quality control process (steps 622 to 626)quantitates the concentration of oligonucleotide in each well. If thisquality control step is performed, an automated liquid handler (2040 ofFIG. 18) is instructed to remove an aliquot from each well of the masterplate and generate a replicate daughter plate for transfer to the UVspectrophotometer (2016 of FIG. 18). The UV spectrophotometer (2016 ofFIG. 18) then measures the optical density of each well at a wavelengthof 260 nanometers. Using standardized conversion factors, amicroprocessor within UV spectrophotometer (2016 of FIG. 18) thencalculates a concentration value from the measured absorbance value foreach well and output the results to Sample Database 520.

[0199] The second illustrative quality control process steps 632 to 636)quantitates the percent of total oligonucleotide in each well that isfull length. If this quality control step is performed, an automatedliquid handler (2040 of FIG. 18) is instructed to remove an aliquot fromeach well of the master plate and generate a replicate daughter platefor transfer to the multichannel capillary gel electrophoresis apparatus(2022 of FIG. 18). The apparatus electrophoretically resolves incapillary tube gels the oligonucleotide product in each well. As theproduct reaches the distal end of the tube gel during electrophoresis, adetection window dynamically measures the optical density of the productthat passes by it. Following electrophoresis, the value of percentproduct that passed by the detection window with respect to time isutilized by a built in microprocessor to calculate the relative sizedistribution of oligonucleotide product in each well. These results arethen output to the Sample Database (520.

[0200] The third illustrative quality control process steps 632 to 636)quantitates the mass of total oligonucleotide in each well that is fulllength. If this quality control step is performed, an automated liquidhandler (2040 of FIG. 2018) is instructed to remove an aliquot from eachwell of the master plate and generate a replicate daughter plate fortransfer to the multichannel liquid electrospray mass spectrometer (2018of FIG. 18). The apparatus then uses electrospray technology to injectthe oligonucleotide product into the mass spectrometer. A built inmicroprocessor calculates the mass-to-charge ratio to arrive at the massof oligonucleotide product in each well. The results are then output toSample Database 520.

[0201] Following completion of the selected quality control processes,the output data is manually examined or is examined using an appropriatealgorithm and a decision is made as to whether or not the plate receives‘Pass’ or ‘Fail’ status. The current criteria for acceptance is that atleast 85% of the oligonucleotides in a multi-well plate must be 85% orgreater full length product as measured by both capillary gelelectrophoresis and mass spectrometry. An input (manual or automated) isthen made into Sample Database 520 as to the pass/fail status of theplate. If a plate fails, the process cycles back to step 500, and a newplate of the same oligonucleotides is automatically placed in the platesynthesis request queue (process 554 of FIG. 15). If a plate receives‘Pass’ status, an automated liquid handler (2040 of FIG. 18) isinstructed to remove appropriate aliquots from each well of the masterplate and generate two replicate daughter plates in which theoligonucleotide in each well is at a concentration of 30 micromolar. Theplate then moves on to process 700 for oligonucleotide activityevaluation.

[0202] 15. Cell Lines for Assaying Oligonucleotide Activity.

[0203] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid, or its gene product, is present at measurablelevels. This can be routinely determined using, for example, PCR orNorthern blot analysis. The following four cell types are provided forillustrative purposes, but other cell types can be routinely used.

[0204] T-24 Cells:

[0205] The transitional cell bladder carcinoma cell line T-24 isobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Life Technologies, Gaithersburg, Md.) supplemented with 10% fetalcalf serum, penicillin 100 units per milliliter, and streptomycin 100micrograms per milliliter (all from Life Technologies). Cells areroutinely passaged by trysinization and dilution when they reach 90%confluence. Cells are routinely seeded into 96-well plates(Falcon-Primaria #3872) at a density of 7000 cells/well for use inRT-PCR analysis. For Northern blotting or other analysis, cells areseeded onto 100 mm or other standard tissue culture plates and treatedsimilarly, using appropriate volumes of medium and oligonucleotide.

[0206] A549 Cells:

[0207] The human lung carcinoma cell line A549 is obtained from the ATCC(Manassas, Va.). A549 cells were routinely cultured in DMEM basal media(Life Technologies) supplemented with 10% fetal calf serum, penicillin100 units per milliliter, and streptomycin 100 micrograms per milliliter(all from Life Technologies). Cells are routinely passaged bytrysinization and dilution when they reach 90% confluence.

[0208] NHDF Cells:

[0209] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corp.) as provided bythe supplier. Cells are maintained for up to 10 passages as recommendedby the supplier.

[0210] HEK Cells:

[0211] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corp. HEKs were routinely maintained in Keratinocyte GrowthMedium (Clonetics Corp.) as provided by the supplier. Cell are routinelymaintained for up to 10 passages as recommended by the supplier.

[0212] 16. Treatment of Cells with Candidate Compounds:

[0213] When cells reach about 80% confluency, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 200 μl Opti-MEM-1™ reduced-serum medium (Life Technologies)and then treated with 130 μl of Opti-MEM-1™ containing 3.75 μg/mlLIPOFECTIN (Life Technologies) and the desired oligonucleotide at afinal concentration of 150 nM. After 4 hours of treatment, the mediumwas replaced with fresh medium. Cells were harvested 16 hours afteroligonucleotide treatment.

[0214] 17. Assaying Oligonucleotide Activity:

[0215] Oligonucleotide-mediated modulation of expression of a targetnucleic acid can be assayed in a variety of ways known in the art. Forexample, target RNA levels can be quantitated by, e.g., Northern blotanalysis, competitive PCR, or reverse transcriptase polymerase chainreaction (RT-PCR). RNA analysis can be performed on total cellular RNAor, preferably in the case of polypeptide-encoding nucleic acids,poly(A)+ mRNA. For RT-PCR, poly(A)+ mRNA is preferred. Methods of RNAisolation are taught in, for example, Ausubel et al. (Short Protocols inMolecular Biology, 2nd Ed., pp. 4-1 to 4-13, Greene PublishingAssociates and John Wiley & Sons, New York, 1992). Northern blotanalysis is routine in the art (Id., pp. 4-14 to 4-29). Reversetranscriptase polymerase chain reaction (RT-PCR) can be convenientlyaccomplished using the commercially available ABI PRISM 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Other methods of PCR are also known inthe art.

[0216] Target protein levels can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), Enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to aprotein encoded by a target nucleic acid can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies,(Aerie Corporation, Birmingham, Mich. or via the internet athttp://www.ANTIBODIES-PROBES.com/), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonal,monospecific (‘antipeptide’) and monoclonal antisera are taught by, forexample, Ausubel et al. (Short Protocols in Molecular Biology, 2nd Ed.,pp. 11-3 to 11-54, Greene Publishing Associates and John Wiley & Sons,New York, 1992).

[0217] Immunoprecipitation methods are standard in the art and aredescribed by, for example, Ausubel et al. (Id., pp. 10-57 to 10-63).Western blot (immunoblot) analysis is standard in the art (Id., pp.10-32 to 10-10-35). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art (Id., pp. 11-5 to 11-17).

[0218] Because it is preferred to assay the compounds of the inventionin a batchwise fashion, i.e., in parallel to the automated synthesisprocess described above, preferred means of assaying are suitable foruse in 96-well plates and with robotic means. Accordingly, automatedRT-PCR is preferred for assaying target nucleic acid levels, andautomated ELISA is preferred for assaying target protein levels.

[0219] The assaying step, general procedure step 700, is described indetail in FIG. 16. After an appropriate cell line is selected at processstep 710, a decision is made at decision step 714 as to whether RT-PCRwill be the only method by which the activity of the compounds isevaluated. In some instances, it is desirable to run alternative assaymethods at process step 718; for example, when it is desired to assesstarget polypeptide levels as well as target RNA levels, an immunoassaysuch as an ELISA is run in parallel with the RT-PCR assays. Preferably,such assays are tractable to semi-automated or robotic means.

[0220] When RT-PCR is used to evaluate the activities of the compounds,cells are plated into multi-well plates (typically, 96-well plates) inprocess step 720 and treated with test or control oligonucleotides inprocess step 730. Then, the cells are harvested and lysed in processstep 740 and the lysates are introduced into an apparatus where RT-PCRis carried out in process step 750. A raw data file is generated, andthe data is downloaded and compiled at step 760. Spreadsheet files withdata charts are generated at process step 770, and the experimental datais analyzed at process step 780. Based on the results, a decision ismade at process step 785 as to whether it is necessary to repeat theassays and, if so, the process begins again with step 720. In any event,data from all the assays on each oligonucleotide are complied andstatistical parameters are automatically determined at process step 790.

[0221] 18. Classification of Compounds Based on Their Activity:

[0222] Following assaying, general procedure step 700, oligonucleotidecompounds are classified according to one or more desired properties.Typically, three classes of compounds are used: active compounds,marginally active (or ‘marginal’) compounds and inactive compounds. Tosome degree, the selection criteria for these classes vary from targetto target, and members of one or more classes may not be present for agiven set of oligonucleotides.

[0223] However, some criteria are constant. For example, inactivecompounds will typically comprise those compounds having 5% or lessinhibition of target expression (relative to basal levels). Activecompounds will typically cause at least 30% inhibition of targetexpression, although lower levels of inhibition are acceptable in someinstances. Marginal compounds will have activities intermediate betweenactive and inactive compounds, with preferred marginal compounds havingactivities more like those of active compounds.

[0224] 19. Optimization of Lead Compounds by Sequence.

[0225] One means by which oligonucleotide compounds are optimized foractivity is by varying their nucleobase sequences so that differentregions of the target nucleic acid are targeted. Some such regions willbe more accessible to oligonucleotide compounds than others, and‘sliding’ a nucleobase sequence along a target nucleic acid only a fewbases can have significant effects on activity. Accordingly, varying oradjusting the nucleobase sequences of the compounds of the invention isone means by which suboptimal compounds can be made optimal, or by whichnew active compounds can be generated.

[0226] The operation of the gene walk process 1100 detailed in steps1104-1112 of FIG. 17 is detailed as follows. As used herein, the term‘gene walk’ is defined as the process by which a specifiedoligonucleotide sequence x that binds to a specified nucleic acid targety is used as a frame of reference around which a series of newoligonucleotides sequences capable of hybridizing to nucleic acid targety are generated that are sequence shifted increments of oligonucleotidesequence x. Gene walking can be done “downstream”, ‘upstream’ or in bothdirections from a specified oligonucleotide.

[0227] During step 1104 the user manually enters the identificationnumber of the oligonucleotide sequence around which it is desired toexecute gene walk process 1100 and the name of the corresponding targetnucleic acid. The user then enters the scope of the gene walk at step1104, by which is meant the number of oligonucleotide sequences that itis desired to generate. The user then enters in step 1108 a positiveinteger value for the sequence shift increment. Once this data isgenerated, the gene walk is effected. This causes a subroutine to beexecuted that automatically generates the desired list of sequences bywalking along the target sequence. At that point, the user proceeds toprocess 400 to assign chemistries to the selected oligonucleotides.

[0228] Example 16 below, details a gene walk. In subsequent steps, thisnew set of nucleobase sequences generated by the gene walk is used todirect the automated synthesis at general procedure step 500 of a secondset of candidate oligonucleotides. These compounds are then takenthrough subsequent process steps to yield active compounds or reiteratedas necessary to optimize activity of the compounds.

[0229] 20. Optimization of Lead Compounds by Chemistry.

[0230] Another means by which oligonucleotide compounds of the inventionare optimized is by reiterating portions of the process of the inventionusing marginal compounds from the first iteration and selectingadditional chemistries to the nucleobase sequences thereof.

[0231] Thus, for example, an oligonucleotide chemistry different fromthat of the first set of oligonucleotides is assigned at generalprocedure step 400. The nucleobase sequences of marginal compounds areused to direct the synthesis at general procedure step 500 of a secondset of oligonucleotides having the second assigned chemistry. Theresulting second set of oligonucleotide compounds is assayed in the samemanner as the first set at procedure process step 700 and the resultsare examined to determine if compounds having sufficient activity havebeen generated at decision step 800.

[0232] 21. Identification of Sites Amenable to Antisense Technologies.

[0233] In a related process, a second oligonucleotide chemistry isassigned at procedure step 400 to the nucleobase sequences of all of theoligonucleotides (or, at least, all of the active and marginalcompounds) and a second set of oligonucleotides is synthesized atprocedure step 500 having the same nucleobase sequences as the first setof compounds. The resulting second set of oligonucleotide compounds isassayed in the same manner as the first set at procedure step 700 andactive and marginal compounds are identified at procedure steps 800 and1000.

[0234] In order to identify sites on the target nucleic acid that areamenable to a variety of antisense technologies, the followingmathematically simple steps are taken. The sequences of active andmarginal compounds from two or more such automated syntheses/assays arecompared and a set of nucleobase sequences that are active, ormarginally so, in both sets of compounds is identified. The reversecomplements of these nucleobase sequences corresponds to sequences ofthe target nucleic acid that are tractable to a variety of antisense andother sequence-based technologies.

[0235] These antisense-sensitive sites are assembled into contiguoussequences (contigs) using the procedures described for assembling targetnucleotide sequences (at procedure step 200).

[0236] 22. Systems for Executing Preferred Methods of the Invention.

[0237] An embodiment of computer, network and instrument resources foreffecting the methods of the invention is shown in FIG. 18. In thisembodiment, four computer servers are provided. First, a large databaseserver 2002 stores all chemical structure, sample tracking and genomic,assay, quality control, and program status data. Further, this databaseserver serves as the platform for a document management system. Second,a compute engine 2004 runs computational programs including RNA folding,oligonucleotide walking, and genomic searching. Third, a file server2006 allows raw instrument output storage and sharing of robotinstructions. Fourth, a groupware server 2008 enhances staffcommunication and process scheduling.

[0238] A redundant high-speed network system is provided between themain servers and the bridges 2026, 2028 and 2030. These bridges providereliable network access to the is many workstations and instrumentsdeployed for this process.

[0239] The instruments selected to support this embodiment are alldesigned to sample directly from standard 96 well microtiter plates, andinclude an optical density reader 2016, a combined liquid chromatographyand mass spectroscopy instrument 2018, a gel fluorescence andscintillation imaging system 2032 and 2042, a capillary gelelectrophoreses system 2022 and a real-time PCR system 2034.

[0240] Most liquid handling is accomplished automatically using robotswith individually controllable robotic pipetters 2038 and 2020 as wellas a 96-well pipette system 2040 for duplicating plates. Windows NT orMacintosh workstations 2044, 2024, and 2036 are deployed for instrumentcontrol, analysis and productivity support.

[0241] 23. Relational Database.

[0242] Data is stored in an appropriate database. For use with themethods of the invention, a relational database is preferred. FIG. 19illustrates the data structure of a sample relational database. Variouselements of data are segregated among linked storage elements of thedatabase.

EXAMPLES

[0243] The following examples illustrate the invention and are notintended to limit the same. Those skilled in the art will recognize, orbe able to ascertain through routine experimentation, numerousequivalents to the specific procedures, materials and devices describedherein. Such equivalents are considered to be within the scope of thepresent invention.

Example 1 Selection of CD40 as a Target

[0244] Cell-cell interactions are a feature of a variety of biologicalprocesses. In the activation of the immune response, for example, one ofthe earliest detectable events in a normal inflammatory response isadhesion of leukocytes to the vascular endothelium, followed bymigration of leukocytes out of the vasculature to the site of infectionor injury. The adhesion of leukocytes to vascular endothelium is anobligate step in their migration out of the vasculature (for a review,see Albelda et al., FASEB J., 1994, 8, 504). As is well known in theart, cell-cell interactions are also critical for propagation of bothB-lymphocytes and T-lymphocytes resulting in enhanced humoral andcellular immune responses, respectively (for a reviews, see Makgoba etal., Immunol. Today, 1989, 10, 417; Janeway, Sci. Amer., 1993, 269, 72).

[0245] CD40 was first characterized as a receptor expressed onB-lymphocytes. It was later found that engagement of B-cell CD40 withCD40L expressed on activated T-cells is essential for T-cell dependentB-cell activation (i.e. proliferation, immunoglobulin secretion, andclass switching) (for a review, see Gruss et al. Leuk. Lymphoma, 1997,24, 393). A full cDNA sequence for CD40 is available (GenBank accessionnumber X60592, incorporated herein as SEQ ID NO:85).

[0246] As interest in CD40 mounted, it was subsequently revealed thatfunctional CD40 is expressed on a variety of cell types other thanB-cells, including macrophages, dendritic cells, thymic epithelialcells, Langerhans cells, and endothelial cells (Ibid.). These studieshave led to the current belief that CD40 plays a much broader role inimmune regulation by mediating interactions of T-cells with cell typesother than B-cells. In support of this notion, it has been shown thatstimulation of CD40 in macrophages and dendritic results is required forT-cell activation during antigen presentation (Id.). Recent evidencepoints to a role for CD40 in tissue inflammation as well. Production ofthe inflammatory mediators IL-12 and nitric oxide by macrophages hasbeen shown to be CD40 dependent (Buhlmann et al., J. Clin. Immunol.,1996, 16, 83). In endothelial cells, stimulation of CD40 by CD40L hasbeen found to induce surface expression of E-selectin, ICAM-1, andVCAM-1, promoting adhesion of leukocytes to sites of inflammation(Buhlmann et al., J. Clin. Immunol, 1996, 16, 83; Gruss et al., LeukLymphoma, 1997, 24, 393). Finally, a number of reports have documentedoverexpression of CD40 in epithelial and hematopoietic tumors as well astumor infiltrating endothelial cells, indicating that CD40 may play arole in tumor growth and/or angiogenesis as well (Gruss et al., LeukLymphoma, 1997, 24, 393-422; Kluth et al. Cancer Res, 1997, 57, 891).

[0247] Due to the pivotal role that CD40 plays in humoral immunity, thepotential exists that therapeutic strategies aimed at downregulatingCD40 may provide a novel class of agents useful in treating a number ofimmune associated disorders, including but not limited to graft versushost disease, graft rejection, and autoimmune diseases such as multiplesclerosis, systemic lupus erythematosus, and certain forms of arthritis.Inhibitors of CD40 may also prove useful as an anti-inflammatorycompound, and could therefore be useful as treatment for a variety ofdiseases with an inflammatory component such as asthma, rheumatoidarthritis, allograft rejections, inflammatory bowel disease, and variousdermatological conditions, including psoriasis. Finally, as more islearned about the association between CD40 overexpression and tumorgrowth, inhibitors of CD40 may prove useful as anti-tumor agents aswell.

[0248] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of CD40. To date, strategies aimed atinhibiting CD40 function have involved the use of a variety of agentsthat disrupt CD40/CD40L binding. These include monoclonal antibodiesdirected against either CD40 or CD40L, soluble forms of CD40, andsynthetic peptides derived from a second CD40 binding protein, A20. Theuse of neutralizing antibodies against CD40 and/or CD40L in animalmodels has provided evidence that inhibition of CD40 stimulation wouldhave therapeutic benefit for GVHD, allograft rejection, rheumatoidarthritis, SLE, MS, and B-cell lymphoma (Buhlmann et al., J. Clin.Immunol, 1996, 16, 83). However, due to the expense, short half-life,and bioavailability problems associated with the use of large proteinsas therapeutic agents, there is a long felt need for additional agentscapable of effectively inhibiting CD40 function. Oligonucleotidescompounds avoid many of the pitfalls of current agents used to blockCD40/CD40L interactions and may therefore prove to be uniquely useful ina number of therapeutic applications.

Example 2 Generation of Virtual Oligonucleotides Targeted to CD40

[0249] The process of the invention was used to select oligonucleotidestargeted to CD40, generating the list of oligonucleotide sequences withdesired properties as shown in FIG. 22. From the assembled CD40sequence, the process began with determining the desired oligonucleotidelength to be eighteen nucleotides, as represented in step 2500. Allpossible oligonucleotides of this length were generated by Oligo 5.0™,as represented in step 2504. Desired thermodynamic properties wereselected in step 2508. The single parameter used was oligonucleotides ofmelting temperature less than or equal to 40° C. were discarded. In step2512, oligonucleotide melting temperatures were calculated by Oligo5.0™. Oligonucleotide sequences possessing an undesirable score werediscarded. It is believed that oligonucleotides with meltingtemperatures near or below physiological and cell culture temperatureswill bind poorly to target sequences. All oligonucleotide sequencesremaining were exported into a spreadsheet. In step 2516, desiredsequence properties are selected. These include discardingoligonucleotides with at least one stretch of four guanosines in a rowand stretches of six of any other nucleotide in a row. In step 2520, aspreadsheet macro removed all oligonucleotides containing the textstring ‘GGGG’. In step 2524, another spreadsheet macro removed alloligonucleotides containing the text strings ‘AAAAAA’ or ‘CCCCCC’ or‘TTTTTT’. From the remaining oligonucleotide sequences, 84 sequenceswere selected manually with the criteria of having an uniformdistribution of oligonucleotide sequences throughout the targetsequence, as represented in step 2528. These oligonucleotide sequenceswere then passed to the next step in the process, assigning actualoligonucleotide chemistries to the sequences.

Example 3 Input Files For Automated Oligonucleotide Synthesis

[0250] Command File (.cmd File)

[0251] Table 2 is a command file for synthesis of oligonucleotide havingregions of 2′-O-(methoxyethyl) nucleosides and region of 2′-deoxynucleosides each linked by phosphorothioate internucleotide linkages.TABLE 2 SOLID_SUPPORT_SKIP BEGIN Next_Sequence END  INITIAL-WASH BEGINAdd ACN 300 Drain 10 END  LOOP-BEGIN  DEBLOCK BEGIN Prime TCA Load TrayRepeat 2 Add TCA 150 Wait 10 Drain 8 End_Repeat Remove Tray Add TCA 125Wait 10 Drain 8 END WASH_AFTER_DEBLOCK BEGIN Repeat 3 Add ACN 250 To_AllDrain 10 End_Repeat END COUPLING BEGIN if class = DEOXY_THIOATE Nozzlewash <act1> prime <act1> prime <seq> Add <act1> 70 + <seq> 70 Wait 40Drain 5 end-if if class = MOE_THIOATE Nozzle wash <act1> Prime <act1>prime <seq> Add <act1> 120 + <seq> 120 Wait 230 Drain 5 End_if ENDWASH_AFTER_COUPLING BEGIN Add ACN 200 To_All Drain 10 END OXIDIZE BEGINif class = DEOXY_THIOATE Add BEAU 180 Wait 40 Drain 7 end_if if class =MOE_THIOATE Add BEAU 200 Wait 120 Drain 7 end_if END CAP BEGIN Add CAP_B80 + CAP_A 80 Wait 20 Drain 7 END WASH_AFTER_CAP BEGIN Add ACN 150To_All Drain 5 Add ACN 250 To_All Drain 11 END BASE_COUNTER BEGINNext_Sequence END LOOP_END DEBLOCK_FINAL BEGIN Prime TCA Load TrayRepeat 2 Add TCA 150 To_All Wait 10 Drain 8 End_Repeat Remove Tray AddTCA 125 To_All Wait 10 Drain 10 END FINAL_WASH BEGIN Repeat 4 Add ACN300 to_All Drain_12 End_Repeat END ENDALL BEGIN Wait 3 END

[0252] Sequence Files (.seq Files)

[0253] Table 3 is a seq file for oligonucleotides having 2′-deoxynucleosides linked by phosphorothioate internucleotide linkages. TABLE 3Identity of columns: Syn #, Well, Scale, Nucleotide at particularposition (identified using base identifier followed by backboneidentifier where ‘s’ is phosphorothioate). Note the columns wrap aroundto next line when longer than one line. 1 A01 200 As Cs Cs As Gs Gs AsCs Gs Gs  Cs  Gs Gs As Cs Cs As Gs 2 A02 200 As Cs Gs Gs Cs Gs Gs As CsCs  As  Gs As Gs Ts Gs Gs As 3 A03 200 As Cs Cs As As Gs Cs As GsAs  Cs  Gs Gs As Gs As Cs Gs 4 A04 200 As Gs Gs As Gs As Cs Cs CsCs  Gs  As Cs Gs As As Cs Gs 5 A05 200 As Cs Cs Cs Cs Gs As Cs GsAs  As  Cs Gs As Cs Ts Gs Gs 6 A06 200 As Cs Gs As As Cs Gs As CsTs  Gs  Gs Cs Gs As Cs As Gs 7 A07 200 As Cs Gs As Cs Ts Gs Gs CsGs  As  Cs As Gs Gs Ts As Gs 8 A08 200 As Cs As Gs Gs Ts As Gs GsTs  Cs  Ts Ts Gs Gs Ts Gs Gs 9 A09 200 As Gs Gs Ts Cs Ts Ts Gs GsTs  Gs  Gs Gs Ts Gs As Cs Gs 10 A10 200 As Gs Ts Cs As Cs Gs As CsAs  As  Gs As As As Cs As Cs 11 A11 200 As Cs Gs As Cs As As Gs AsAs  As  Cs As Cs Gs Gs Ts Cs 12 A12 200 As Gs As As As Cs As Cs GsGs  Ts  Cs Gs Gs Ts Cs Cs Ts 13 B01 200 As As Cs As Cs Gs Gs Ts CsGs  Gs  Ts Cs Cs Ts Gs Ts Cs 14 B02 200 As Cs Ts Cs As Cs Ts Gs AsCs  Gs  Ts Gs Ts Cs Ts Cs As 15 B03 200 As Cs Gs Gs As As Gs Gs AsAs  Cs  Gs Cs Cs As Cs Ts Ts 16 B04 200 As Ts Cs Ts Gs Ts Gs Gs AsCs  Cs  Ts Ts Gs Ts Cs Ts Cs 17 B05 200 As Cs As Cs Ts Ts Cs Ts TsCs  Cs  Gs As Cs Cs Gs Ts Gs 18 B06 200 As Cs Ts Cs Ts Cs Gs As CsAs  Cs  As Gs Gs As Cs Gs Ts 19 B07 200 As As As Cs Cs Cs Cs As GsTs  Ts  Cs Gs Ts Cs Ts As As 20 B08 200 As Ts Gs Ts Cs Cs Cs Cs AsAs  As  Gs As Cs Ts As Ts Gs 21 B09 200 As Cs Gs Cs Ts Cs Gs Gs GsAs  Cs  Gs Gs Gs Ts Cs As Gs 22 B10 200 As Gs Cs Cs Gs As As Gs AsAs  Gs  As Gs Gs Ts Ts As Cs 23 B11 200 As Cs As Cs As Gs Ts As GsAs  Cs  Gs As As As Gs Cs Ts 24 B12 200 As Cs As Cs Ts Cs Ts Gs GsTs  Ts  Ts Cs Ts Gs Gs As Cs 25 C01 200 As Cs Gs As Cs Cs As Gs AsAs  As  Ts As Gs Ts Ts Ts Ts 26 C02 200 As Gs Ts Ts As As As As GsGs  Gs  Cs Ts Gs Cs Ts As Gs 27 C03 200 As Gs Gs Ts Ts Gs Ts Gs AsCs  Gs  As Cs Gs As Gs Gs Ts 28 C04 200 As As Ts Gs Ts As Cs Cs TsAs  Cs  Gs Gs Ts Ts Gs Gs Cs 29 C05 200 As Gs Ts Cs As Cs Gs Ts CsCs  Ts  Cs Ts Cs Ts Gs Ts Cs 30 C06 200 Cs Ts Gs Gs Cs Gs As Cs AsGs  Gs  Ts As Gs Gs Ts Cs Ts 31 C07 200 Cs Ts Cs Ts Gs Ts Gs Ts GsAs  Cs  Gs Gs Ts Gs Gs Ts Cs 32 C08 200 Cs As Gs Gs Ts Cs Gs Ts CsTs  Ts  Cs Cs Cs Gs Ts Gs Gs 33 C09 200 Cs Ts Gs Ts Gs Gs Ts As GsAs  Cs  Gs Ts Gs Gs As Cs As 34 C10 200 Cs Ts As As Cs Gs As Ts GsTs  Cs  Cs Cs Cs As As As Gs 35 C11 200 Cs Ts Gs Ts Ts Cs Gs As CsAs  Cs  Ts Cs Ts Gs Gs Ts Ts 36 C12 200 Cs Ts Gs Gs As Cs Cs As AsCs  As  Cs Gs Ts Ts Gs Ts Cs 37 D01 200 Cs Cs Gs Ts Cs Cs Gs Ts GsTs  Ts  Ts Gs Ts Ts Cs Ts Gs 38 D02 200 Cs Ts Gs As Cs Ts As Cs AsAs  Cs  As Gs As Cs As Cs Cs 39 D03 200 Cs As As Cs As Gs As Cs AsCs  Cs  As Gs Gs Gs Gs Ts Cs 40 D04 200 Cs As Gs Gs Gs Gs Ts Cs CsTs  As  Gs Cs Cs Gs As Cs Ts 41 D05 200 Cs Ts Cs Ts As Gs Ts Ts AsAs  As  As Gs Gs Gs Cs Ts Gs 42 D06 200 Cs Ts Gs Cs Ts As Gs As AsGs  Gs  As Cs Cs Gs As Gs Gs 43 D07 200 Cs Ts Gs As As As Ts Gs TsAs  Cs  Cs Ts As Cs Gs Gs Ts 44 D08 200 Cs As Cs Cs Cs Gs Ts Ts TsGs  Ts  Cs Cs Gs Ts Cs As As 45 D09 200 Cs Ts Cs Gs As Ts As Cs GsGs  Gs  Ts Cs As Gs Ts Cs As 46 D10 200 Gs Gs Ts As Gs Gs Ts Cs TsTs  Gs  Gs Ts Gs Gs Gs Ts Gs 47 D11 200 Gs As Cs Ts Ts Ts Gs Cs CsTs  Ts  As Cs Gs Gs As As Gs 48 D12 200 Gs Ts Gs Gs As Gs Ts Cs TsTs  Ts  Gs Ts Cs Ts Gs Ts Gs 49 E01 200 Gs Gs As Gs Ts Cs Ts Ts TsGs  Ts  Cs Ts Gs Ts Gs Gs Ts 50 E02 200 Gs Gs As Cs As Cs Ts Cs TsCs  Gs  As Cs As Cs As Gs Gs 51 E03 200 Gs As Cs As Cs As Gs Gs AsCs  Gs  Ts Gs Gs Cs Gs As Gs 52 E04 200 Gs As Gs Ts As Cs Gs As GsCs  Gs  Gs Gs Cs Cs Gs As As 53 E05 200 Gs As Cs Ts As Ts Gs Gs TsAs  Gs  As Cs Gs Cs Ts Cs Gs 54 E06 200 Gs As As Gs As Gs Gs Ts TsAs  Cs  As Cs As Gs Ts As Gs 55 E07 200 Gs As Gs Gs Ts Ts As Cs AsCs  As  Gs Ts As Gs As Cs Gs 56 E08 200 Gs Ts Ts Gs Ts Cs Cs Gs TsCs  Cs  Gs Ts Gs Ts Ts Ts Gs 57 E09 200 Gs As Cs Ts Cs Ts Cs Gs GsGs  As  Cs Cs As Cs Cs As Cs 55 E10 200 Gs Ts As Gs Gs As Gs As AsCs  Cs  As Cs Gs As Cs Cs As 59 E11 200 Gs Gs Ts Ts Cs Ts Ts Cs GsGs  Ts  Ts Gs Gs Ts Ts As Ts 60 E12 200 Gs Ts Gs Gs Gs Gs Ts Ts CsGs  Ts  Cs Cs Ts Ts Gs Gs Gs 61 F01 200 Gs Ts Cs As Cs Gs Ts Cs CsTs  Cs  Ts Gs As As As Ts Gs 62 F02 200 Gs Ts Cs Cs Ts Cs Cs Ts AsCs  Cs  Gs Ts Ts Ts Cs Ts Cs 63 F03 200 Gs Ts Cs Cs Cs Cs As Cs GsTs  Cs  Cs Gs Ts Cs Ts Ts Cs 64 F04 200 Ts Cs As Cs Cs As Gs Gs AsCs  Gs  Gs Cs Gs Gs As Cs Cs 65 F05 200 Ts As Cs Cs As As Gs Cs AsGs  As  Cs Gs Gs As Gs As Cs 66 F06 200 Ts Cs Cs Ts Gs Ts Cs Ts TsTs  Gs  As Cs Cs As Cs Ts Cs 67 F07 200 Ts Gs Ts Cs Ts Ts Ts Gs AsCs  Cs  As Cs Ts Cs As Cs Ts 68 F08 200 Ts Gs As Cs Cs As Cs Ts CsAs  Cs  Ts Gs As Cs Gs Ts Gs 69 F09 200 Ts Gs As Cs Gs Ts Gs Ts CsTs  Cs  As As Gs Ts Gs As Cs 70 F10 200 Ts Cs As As Gs Ts Gs As CsTs  Ts  Ts Gs Cs Cs Ts Ts As 71 F11 200 Ts Gs Ts Ts Ts As Ts Gs AsCs  Gs  Cs Ts Gs Gs Gs Gs Ts 72 F12 200 Ts Ts As Ts Gs As Cs Gs CsTs  Gs  Gs Gs Gs Ts Ts Gs Gs 73 G01 200 Ts Gs As Cs Gs Cs Ts Gs GsGs  Gs  Ts Ts Gs Gs As Ts Cs 74 G02 200 Ts Cs Gs Ts Cs Ts Ts Cs CsCs  Gs  Ts Gs Gs As Gs Ts Cs 75 G03 200 Ts Gs Gs Ts As Gs As Cs GsTs  Gs  Gs As Cs As Cs Ts Ts 76 G04 200 Ts Ts Cs Ts Ts Cs Cs Gs AsCs  Cs  Gs Ts Gs As Cs As Ts 77 G05 200 Ts Gs Gs Ts As Gs As Cs GsCs  Ts  Cs Gs Gs Gs As Cs Gs 78 G06 200 Ts As Gs As Cs Gs Cs Ts CsGs  Gs  Gs As Cs Gs Gs Gs Ts 79 G07 200 Ts Ts Ts Ts As Cs As Gs TsGs  Gs  Gs As As Cs Cs Ts Gs 80 G08 200 Ts Gs Gs Gs As As Cs Cs TsGs  Ts  Ts Cs Gs As Cs As Cs 81 G09 200 Ts Cs Gs Gs Gs As Cs Cs AsCs  Cs  As Cs Ts As Gs Gs Gs 82 G10 200 Ts As Gs Gs As Cs As As AsCs  Gs  Gs Ts As Gs Gs As Gs 83 G11 200 Ts Gs Cs Ts As Gs As As GsGs  As  Cs Cs Gs As Gs Gs Ts 84 G12 200 Ts Cs Ts Gs Ts Cs As Cs TsCs  Cs  Gs As Cs Gs Ts Gs Gs

[0254] Table 4 is a seq file for oligonucleotides having regions of2′-O-(methoxyethyl)nucleosides and region of 2′-deoxy nucleosides eachlinked by phosphorothioate internucleotide linkages. TABLE 4 Identity ofcolumns: Syn #, Well, Scale, Nucleotide at particular position(identified using base identifier followed by backbone identifier where‘s’ is phosphorothioate and ‘moe’ indicated a 2′-O-(methoxyethy)substituted nucleoside). The columns wrap around to next line whenlonger than one line. 1 A01 200 moeAs   moeCs   moeCs   moeAs   GsGs  As  Cs   Gs   Gs   Cs   Gs   Gs   As   moeCs moeCs    moeAs    moeGs2 A02 200 moeAs   moeCs   moeGs   moeGs   CsGs  Gs  As   Cs   Cs   As   Gs   As   Gs   moeTs moeGs    moeGs    moeAs3 A03 200 moeAs   moeCs   moeCs   moeAs   AsGs  Cs  As   Gs   As   Cs   Gs   Gs   As   moeGs moeAs    moeCs    moeGs4 A04 200 moeAs   moeGs   moeGs   moeAs   GsAs  Cs  Cs   Cs   Cs   Gs   As   Cs   Gs   moeAs moeAs    moeCs    moeGs5 A05 200 moeAs   moeCs   moeCs   moeCs   CsGs  As  Cs   Gs   As   As   Cs   Gs   As   moeCs moeTs    moeGs   moeGs6 A06 200 moeAs   moeCs   moeGs   moeAs   AsCs  Gs  As   Cs   Ts   Gs   Gs   Cs   Gs   moeAs moeCs    moeAs    moeGs7 A07 200 moeAs   moeCs   moeGs   moeAs   CsTs  Gs  Gs   Cs   Gs   As   Cs   As   Gs   moeGs moeTs    moeAs    moeGs8 A08 200 moeAs   moeCs   moeAs   moeGs   GsTs  As  Gs   Gs   Ts   Cs   Ts   Ts   Gs   moeGs moeTs    moeGs    moeGs9 A09 200 moeAs   moeGs   moeGs   moeTs   CsTs  Ts  Gs   Gs   Ts   Gs   Gs   Gs   Ts   moeGs moeAs    moeCs    moeGs10 A10 200 moeAs   moeGs   moeTs   moeCs   AsCs  Gs  As   Cs   As   As   Gs   As   As   moeAs moeCs    moeAs    moeCs11 A11 200 moeAs   moeCs   moeGs   moeAs   CsAs  As  Gs   As   As   As   Cs   As   Cs   moeGs moeGs    moeTs    moeCs12 A12 200 moeAs   moeGs   moeAs   moeAs   As Cs  As  Cs   Gs   Gs   Ts   Cs   Gs   Gs   moeTs moeCs    moeCs    moeTs 13 B01 200moeAs   moeAs   moeCs   moeAs   CsGs  Gs  Ts   Cs   Gs   Gs   Ts   Cs   Cs   moeTs moeGs    moeTs    moeCs14 B02 200 moeAs   moeCs   moeTs   moeCs   AsCs  Ts  Gs   As   Cs   Gs   Ts   Gs   Ts   moeCs moeTs    moeCs    moeAs15 B03 200 moeAs   moeCs   moeGs   moeGs   AsAs  Gs  Gs   As   As   Cs   Gs   Cs   Cs   moeAs moeCs    moeTs    moeTs16 B04 200 moeAs   moeTs   moeCs   moeTs   GsTs  Gs  Gs   As   Cs   Cs   Ts   Ts   Gs   moeTs moeCs    moeTs    moeCs17 B05 200 moeAs   moeCs   moeAs   moeCs   TsTs  Cs  Ts   Ts   Cs   Cs   Gs   As   Cs   moeCs moeGs    moeTs    moeGs18 206 200 moeAs   moeCs   moeTs   moeCs   TsCs  Gs  As   Cs   As   Cs   As   Gs   Gs   moeAs moeCs    moeGs    moeTs19 B07 200 moeAs   moeAs   moeAs   moeCs   CsCs  Cs  As   Gs   Ts   Ts   Cs   Gs   Ts   moeCs moeTs    moeAs    moeAs20 B08 200 moeAs   moeTs   moeGs   moeTs   CsCs  Cs  Cs   As   As   As   Gs   As   Cs   moeTs moeAs    moeTs    moeGs21 B09 200 moeAs   moeCs   moeGs   moeCs   TsCs  Gs  Gs   Gs   As   Cs   Gs   Gs   Gs   moeTs moeCs    moeAs    moeGs22 B10 200 moeAs   moeGs   moeCs   moeCs   GsAs  As  Gs   As   As   Gs   As   Gs   Gs   moeTs moeTs    moeAs    moeCs23 B11 200 moeAs   moeCs   moeAs   moeCs   AsGs  Ts  As   Gs   As   Cs   Gs   As   As   moeAs moeGs    moeCs    moeTs24 B12 200 moeAs   moeCs   moeAs   moeCs   TsCs  Ts  Gs   Gs   Ts   Ts   Ts   Cs   Ts   moeGs moeGs    moeAs    moeCs25 C01 200 moeAs   moeCs   moeGs   moeAs   CsCs  As  Gs   As   As   As   Ts   As   Gs   moeTs moeTs    moeTs    moeTs26 C02 200 moeAs   moeGs   moeTs   moeTs   AsAs  As  As   Gs   Gs   Gs   Cs   Ts   Gs   moeCs moeTs    moeAs    moeGs27 C03 200 moeAs   moeGs   moeGs   moeTs   TsGs  Ts  Gs   As   Cs   Gs   As   Cs   Gs   moeAs moeGs    moeGs    moeTs28 C04 200 moeAs   moeAs   moeTs   moeGs   TsAs  Cs  Cs   Ts   As   Cs   Gs   Gs   Ts   moeTs moeGs    moeGs    moeCs29 C05 200 moeAs   moeGs   moeTs   moeCs   AsCs  Gs  Ts   Cs   Cs   Ts   Cs   Ts   Cs   moeTs moeGs    moeTs    moeCs30 C06 200 moeCs   moeTs   moeGs   moeGs   CsGs  As  Cs   As   Gs   Gs   Ts   As   Gs   moeGs moeTs    moeCs    moeTs31 C07 200 moeCs   moeTs   moeCs   moeTs   GsTs  Gs  Ts   Gs   As   Cs   Gs   Gs   Ts   moeGs moeGs    moeTs    moeCs32 C08 200 moeCs   moeAs   moeGs   moeGs   TsCs  Gs  Ts   Cs   Ts   Ts   Cs   Cs   Cs   moeGs moeTs    moeGs    moeGs33 C09 200 moeCs   moeTs   moeGs   moeTs   GsGs  Ts  As   Gs   As   Cs   Gs   Ts   Gs   moeGs moeAs    moeCs    moeAs34 C10 200 moeCs   moeTs   moeAs   moeAs   CsGs  As  Ts   Gs   Ts   Cs   Cs   Cs   Cs   moeAs moeAs    moeAs    moeGs35 C11 200 moeCs   moeTs   moeGs   moeTs   TsCs  Gs  As   Cs   As   Cs   Ts   Cs   Ts   moeGs moeGs    moeTs    moeTs36 C12 200 moeCs   moeTs   moeGs   moeGs   AsCs  Cs  As   As   Cs   As   Cs   Gs   Ts   moeTs moeGs    moeTs    moeCs37 D01 200 moeCs   moeCs   moeGs   moeTs   CsCs  Gs  Ts   Gs   Ts   Ts   Ts   Gs   Ts   moeTs moeCs    moeTs    moeGs38 D02 200 moeCs   moeTs   moeGs   moeAs   CsTs  As  Cs   As   As   Cs   As   Gs   As   moeCs moeAs    moeCs    moeCs39 D03 200 moeCs   moeAs   moeAs   moeCs   AsGs  As  Cs   As   Cs   Cs   As   Gs   Gs   moeGs moeGs    moeTs    moeCs40 D04 200 moeCs   moeAs   moeGs   moeGs   GsGs  Ts  Cs   Cs   Ts   As   Gs   Cs   Cs   moeGs moeAs    moeCs    moeTs41 D05 200 moeCs   moeTs   moeCs   moeTs   AsGs  Ts  Ts   As   As   As   As   Gs   Gs   moeGs moeCs    moeTs    moeGs42 D06 200 moeCs   moeTs   moeGs   moeCs   TsAs  Gs  As   As   Gs   Gs   As   Cs   Cs   moeGs moeAs    moeGs    moeGs43 D07 200 moeCs   moeTs   moeGs   moeAs   AsAs  Ts  Gs   Ts   As   Cs   Cs   Ts   As   moeCs moeGs    moeGs    moeTs44 D08 200 moeCs   moeAs   moeCs   moeCs   CsGs  Ts  Ts   Ts   Gs   Ts   Cs   Cs   Gs   moeTs moeCs    moeAs    moeAs45 D09 200 moeCs   moeTs   moeCs   moeGs   AsTs  As  Cs   Gs   Gs   Gs   Ts   Cs   As   moeGs moeTs    moeCs    moeAs46 D10 200 moeGs   moeGs   moeTs   moeAs   GsGs  Ts  Cs   Ts   Ts   Gs   Gs   Ts   Gs   moeGs moeGs    moeTs    moeGs47 D11 200 moeGs   moeAs   moeCs   moeTs   TsTs  Gs  Cs   Cs   Ts   Ts   As   Cs   Gs   moeGs moeAs    moeAs    moeGs48 D12 200 moeGs   moeTs   moeGs   moeGs   AsGs  Ts  Cs   Ts   Ts   Ts   Gs   Ts   Cs   moeTs moeGs    moeTs    moeGs49 E01 200 moeGs   moeGs   moeAs   moeGs   TsCs  Ts  Ts   Ts   Gs   Ts   Cs   Ts   Gs   moeTs moeGs    moeGs    moeTs50 E02 200 moeGs   moeGs   moeAs   moeCs   AsCs  Ts  Cs   Ts   Cs   Gs   As   Cs   As   moeCs moeAs    moeGs    moeGs51 E03 200 moeGs   moeAs   moeCs   moeAs   CsAs  Gs  Gs   As   Cs   Gs   Ts   Gs   Gs   moeCs moeGs    moeAs    moeGs52 E04 200 moeGs   moeAs   moeGs   moeTs   AsCs  Gs  As   Gs   Cs   Gs   Gs   Gs   Cs   moeCs moeGs    moeAs    moeAs53 E05 200 moeGs   moeAs   moeCs   moeTs   AsTs  Gs  Gs   Ts   As   Gs   As   Cs   Gs   moeCs moeTs    moeCs    moeGs54 E06 200 moeGs   moeAs   moeAs   moeGs   AsGs  Gs  Ts   Ts   As   Cs   As   Cs   As   moeGs moeTs    moeAs    moeGs55 E07 200 moeGs   moeAs   moeGs   moeGs   TsTs  As  Cs   As   Cs   As   Gs   Ts   As   moeGs moeAs    moeCs    moeGs56 E08 200 moeGs   moeTs   moeTs   moeGs   TsCs  Cs  Gs   Ts   Cs   Cs   Gs   Ts   Gs   moeTs moeTs    moeTs    moeGs57 E09 200 moeGs   moeAs   moeCs   moeTs   CsTs  Cs  Gs   Gs   Gs   As   Cs   Cs   As   moeCs moeCs    moeAs    moeCs58 E10 200 moeGs   moeTs   moeAs   moeGs   GsAs  Gs  As   As   Cs   Cs   As   Cs   Gs   moeAs moeCs    moeCs    moeAs59 E11 200 moeGs   moeGs   moeTs   moeTs   CsTs  Ts  Cs   Gs   Gs   Ts   Ts   Gs   Gs   moeTs moeTs    moeAs    moeTs60 E12 200 moeGs   moeTs   moeGs   moeGs   GsGs  Ts  Ts   Cs   Gs   Ts   Cs   Cs   Ts   moeTs moeGs    moeGs    moeGs61 F01 200 moeGs   moeTs   moeCs   moeAs   CsGs  Ts  Cs   Cs   Ts   Cs   Ts   Gs   As   moeAs moeAs    moeTs    moeGs62 F02 200 moeGs   moeTs   moeCs   moeCs   TsCs  Cs  Ts   As   Cs   Cs   Gs   Ts   Ts   moeTs moeCs    moeTs    moeCs63 F03 200 moeGs   moeTs   moeCs   moeCs   CsCs  As  Cs   Gs   Ts   Cs   Cs   Gs   Ts   moeCs moeTs    moeTs    moeCs64 F04 200 moeTs   moeCs   moeAs   moeCs   CsAs  Gs  Gs   As   Cs   Gs   Gs   Cs   Gs   moeGs moeAs    moeCs    moeCs65 F05 200 moeTs   moeAs   moeCs   moeCs   AsAs  Gs  Cs   As   Gs   As   Cs   Gs   Gs   moeAs moeGs    moeAs    moeCs66 F06 200 moeTs   moeCs   moeCs   moeTs   GsTs  Cs  Ts   Ts   Ts   Gs   As   Cs   Cs   moeAs moeCs    moeTs    moeCs67 F07 200 moeTs   moeGs   moeTs   moeCs   TsTs  Ts  Gs   As   Cs   Cs   As   Cs   Ts   moeCs moeAs    moeCs    moeTs68 F08 200 moeTs   moeGs   moeAs   moeCs   CsAs  Cs  Ts   Cs   As   Cs   Ts   Gs   As   moeCs moeGs    moeTs    moeGs69 F09 200 moeTs   moeGs   moeAs   moeCs   GsTs  Gs  Ts   Cs   Ts   Cs   As   As   Gs   moeTs moeGs    moeAs    moeCs70 F10 200 moeTs   moeCs   moeAs   moeAs   GsTs  Gs  As   Cs   Ts   Ts   Ts   Gs   Cs   moeCs moeTs    moeTs    moeAs71 F11 200 moeTs   moeGs   moeTs   moeTs   TsAs  Ts  Gs   As   Cs   Gs   Cs   Ts   Gs   moeGs moeGs    moeGs    moeTs72 F12 200 moeTs   moeTs   moeAs   moeTs   GsAs  Cs  Gs   Cs   Ts   Gs   Gs   Gs   Gs   moeTs moeTs    moeGs    moeGs73 G01 200 moeTs   moeGs   moeAs   moeCs   GsCs  Ts  Gs   Gs   Gs   Gs   Ts   Ts   Gs   moeGs moeAs    moeTs    moeCs74 G02 200 moeTs   moeCs   moeGs   moeTs   CsTs  Ts  Cs   Cs   Cs   Gs   Ts   Gs   Gs   moeAs moeGs    moeTs    moeCs75 G03 200 moeTs   moeGs   moeGs   moeTs   AsGs  As  Cs   Gs   Ts   Gs   Gs   As   Cs   moeAs moeCs    moeTs    moeTs76 G04 200 moeTs   moeTs   moeCs   moeTs   TsCs  Cs  Gs   As   Cs   Cs   Gs   Ts   Gs   moeAs moeCs    moeAs    moeTs77 G05 200 moeTs   moeGs   moeGs   moeTs   AsGs  As  Cs   Gs   Cs   Ts   Cs   Gs   Gs   moeGs moeAs    moeCs    moeGs78 G06 200 moeTs   moeAs   moeGs   moeAs   CsGs  Cs  Ts   Cs   Gs   Gs   Gs   As   Cs   moeGs moeGs    moeGs    moeTs79 G07 200 moeTs   moeTs   moeTs   moeTs   AsCs  As  Gs   Ts   Gs   Gs   Gs   As   As   moeCs moeCs    moeTs    moeGs80 G08 200 moeTs   moeGs   moeGs   moeGs   AsAs  Cs  Cs   Ts   Gs   Ts   Ts   Cs   Gs   moeAs moeCs    moeAs    moeCs81 G09 200 moeTs   moeCs   moeGs   moeGs   GsAs  Cs  Cs   As   Cs   Cs   As   Cs   Ts   moeAs moeGs    moeGs    moeGs82 G10 200 moeTs   moeAs   moeGs   moeGs   AsCs  As  As   As   Cs   Gs   Gs   Ts   As   moeGs moeGs    moeAs    moeGs83 G11 200 moeTs   moeGs   moeCs   moeTs   AsGs  As  As   Gs   Gs   As   Cs   CsGs   moeAs moeGs    moeGs    moeTs 84G12 200 moeTs   moeCs   moeTs   moeGs   TsCs  As  Cs   Ts   Cs   Cs   Gs   As   Cs   moeGs moeTs    moeGs    moeGs

[0255] Reagent File (.tab File)

[0256] Table 5 is a tab for reagents necessary for synthesizing anoligonucleotides having both 2′-O-(methoxy-ethy)nucleosides and 2′-deoxynucleosides located therein. TABLE 5 Identity of columns: GroupName,Bottle ID, ReagentName, FlowRate, Concentration. Wherein reagent name isidentified using base identifier, ‘moe’ indicated a 2′-O- (methoxyethy)substituted nucleoside and ‘cpg’ indicates a control pore glass solidsupport medium. The columns wrap around to next line when longer thanone line. SUPPORT BEGIN  0 moeG moeGcpg 100 1  0 moe5meC moe5meCcpg 1001  0 moeA moeAcpg 100 1  0 moeT moeTcpg 100 1 END DEBLOCK BEGIN 70 TCATCA 100 1 END WASH BEGIN 65 ACN ACN 190 1 END OXIDIZERS BEGIN 68 BEAUBEAUCAGE 320 1 END CAPPING BEGIN 66 CAP_B CAP_B 220 1 67 CAP_A CAP_A 2301 END DEOXY THIOATE BEGIN 31, 32 Gs deoxyG 270 1 39, 40 5meCs5methyldeoxyC 270 1 37, 38 As deoxyA 270 1 29, 30 Ts deoxyT 270 1 ENDMOE-THIOATE BEGIN 15, 16 moeGs methoxyethoxyG 240 1 23, 24 moe5meCsmethoxyethoxyC 240 1 21, 22 moeAs methoxyethoxyA 240 1 13, 14 moeTsmethoxyethoxyT 240 1 END ACTIVATORS BEGIN 5, 6, 7, 8 SET s-ethyl-tet 280Activates DEOXY_THIOATE MOE_THIOATE END

Example 4 Oligonucleotide Synthesis—96 Well Plate Format

[0257] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry using a multi well automated synthesizerutilizing input files as described in EXAMPLE 3 above. Theoligonucleotides were synthesized by assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE/ABI, Pharmacia). Non-standard nucleosidesare synthesized as per known literature or patented methods. They areutilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0258] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 5 Alternative Oligonucleotide Synthesis

[0259] Unsubstituted and substituted phosphodiester oligo-nucleotidesare alternately synthesized on an automated DNA synthesizer (AppliedBiosystems model 380B) using standard phosphoramidite chemistry withoxidation by iodine.

[0260] Phosphorothioates are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hr), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

[0261] Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0262] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0263] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0264] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, hereby incorporatedby reference.

[0265] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively).

[0266] 3′,-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0267] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0268] Boranophosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

[0269] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedi-methylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleo-sides,as well as mixed backbone compounds having, for instance, alternatingMMI and PO or PS linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0270] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0271] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 6 PNA Synthesis

[0272] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5. They may also be prepared in accordance with U.S.Pat. Nos. 5,539,082; 5,700,922, and 5,719,262, herein incorporated byreference.

Example 7 Chimeric Oligonucleotide Synthesis

[0273] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the ‘gap’ segment oflinked nucleosides is positioned between 5′ and 3′ ‘wing’ segments oflinked nucleosides and a second ‘open end’ type wherein the ‘gap’segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas ‘gapmers’ or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as ‘hemimers’ or ‘wingmers.’

[0274] A. [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0275] Chimeric oligonucleotides having 2′-0-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidites for the DNAportion and 5′-dimethoxytrityl-2′-O-methyl-3′-0-phosphoramidites for 5′and 3′ wings. The standard synthesis cycle is modified by increasing thewait step after the delivery of tetrazole and base to 600 s repeatedfour times for DNA and twice for 2′-0-methyl. The fully protectedoligonucleotide was cleaved from the support and the phosphate group isdeprotected in 3:1 Ammonia/Ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is done to deprotect all bases and the samples areagain lyophilized to dryness.

[0276] B. [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0277] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides are prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0278] C. [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotide

[0279] [2′-0-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites in the wingportions. Sulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1dioxide (Beaucage Reagent) is used to generate the phosphorothioateinternucleotide linkages within the wing portions of the chimericstructures. Oxidization with iodine is used to generate thephosphodiester internucleotide linkages for the center gap.

[0280] Other chimeric oligonucleotides, chimeric oligo-nucleosides andmixed chimeric oligonucleotides/oligo-nucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 8 Output Oligonucleotides from Automated OligonucleotideSynthesis

[0281] Using the seq files, the .cmd files and tab file of Example 3,oligonucleotides were prepared as per the protocol of the 96 well formatof Example 4. The oligonucleotides were prepared utilizingphosphorothioate chemistry to give in one instance a first library ofphosphorothioate oligodeoxynucleotides. The oligonucleotides wereprepared in a second instance as a second library of hybridoligonucleotides having phosphorothioate backbones with a first andthird ‘wing’ region of 2′-O-(methoxyethyl)nucleotides on either side ofa center gap region of 2′-deoxy nucleotides. The two libraries containedthe same set of oligonucleotide sequences. Thus the two libraries areredundant with respect to sequence but are unique with respect to thecombination of sequence and chemistry. Because the sequences of thesecond library of compounds is the same as the first (however thechemistry is different), for brevity sake, the second library is notshown.

[0282] For illustrative purposes Tables 6-a and 6-b show the sequencesof an intial first library, i.e., a library of phosphoroethioateoligonucleotides targeted to a CD40 target. The compounds of Table 6-ashows the members of this listed in compliance with the established rulefor listing SEQ ID NO:, i.e., in numerical SEQ ID NO: order. TABLE 6-aSequences of Oligonucleotides Targeted to CD40 by SEQ ID NO.: SEQ IDNUCLEOBASE SEQUENCE NO. CCAGGCGGCAGGACCACT 1 GACCAGGCGGCAGGACCA 2AGGTGAGACCAGGCGGCA 3 CAGAGGCAGACGAACCAT 4 GCAGAGGCAGACGAACCA 5GCAAGCAGCCCCAGAGGA 6 GGTCAGCAAGCAGCcCCA 7 GACAGCGGTCAGCAAGCA 8GATGGACAGCGGTCAGCA 9 TCTGGATGGACAGCGGTC 10 GGTGGTTCTGGATGGACA 11GTGGGTGGTTCTGGATGG 12 GCAGTGGGTGGTTCTGGA 13 CACAAAGAACAGCACTGA 14CTGGCACAAAGAACAGCA 15 TCCTGGCTGGCACAAAGA 16 CTGTCCTGGCTGGCACAA 17CTCACCAGTTTCTGTCCT 18 TCACTCACCAGTTTCTGT 19 GTGCAGTCACTCACCAGT 20ACTCTGTGCAGTCACTCA 21 CAGTGAACTCTGTGCAGT 22 ATTCCGTTTCAGTGAACT 23GAAGGCATTCCGTTTCAG 24 TTCACCGCAAGGAAGGCA 25 CTCTGTTCCAGGTGTCTA 26CTGGTGGCAGTGTGTCTC 27 TGGGGTCGCAGTATTTGT 28 GGTTGGGGTCGCAGTATT 29CTAGGTTGGGGTCGCAGT 30 GGTGCCCTTCTGCTGGAC 31 CTGAGGTGCCCTTCTGCT 32GTGTCTGTTTCTGAGGTG 33 TGGTGTCTGTTTCTGAGG 34 ACAGGTGCAGATGGTGTC 35TTCACAGGTGCAGATGGT 36 GTGCCAGCCTTCTTCACA 37 TACAGTGCCAGCCTTCTT 38GGACACAGCTCTCACAGG 39 TGCAGGACACAGCTCTCA 40 GAGCGGTGCAGGACACAG 41AAGCCGGGCGAGCATGAG 42 AATCTGCTTGACCCCAAA 43 GAAACCCCTGTAGCAATC 44GTATCAGAAACCCCTGTA 45 GCTCGCAGATGGTATCAG 46 GCAGGGCTCGCAGATGGT 47TGGGCAGGGCTCGCAGAT 48 GACTGGGCAGGGCTCGCA 49 CATTGGAGAAGAAGCCGA 50GATGACACATTGGAGAAG 51 GCAGATGACACATTGGAG 52 TCGAAAGCAGATGACACA 53GTCCAAGGGTGACATTTT 54 CACAGCTTGTCCAAGGGT 55 TTGGTCTCACAGCTTGTC 56CAGGTCTTTGGTCTCACA 57 CTGTTGCACAACCAGGTC 58 GTTTGTGCCTGCCTGTTG 59GTCTTGTTTGTGCCTGCC 60 CCACAGACAACATCAGTC 61 CTGGGGACCACAGACAAC 62TCAGCCGATCCTGGGGAC 63 CACCACCAGGGCTCTCAG 64 GGGATCACCACCAGGGCT 65GAGGATGGCAAACAGGAT 66 ACCAGCACCAAGAGGATG 67 TTTTGATAAAGACCAGCA 68TATTGGTTGGCTTCTTGG 69 GGGTTCCTGCTTGGGGTG 70 GTCGGGAAAATTGATCTC 71GATCGTCGGGAAAATTGA 72 GGAGCCAGGAAGATCGTC 73 TGGAGCCAGGAAGATCGT 74TGGAGCAGCAGTGTTGGA 75 GTAAAGTCTCCTGCACTG 76 TGGCATCCATGTAAAGTC 77CGGTTGGCATCCATGTAA 78 CTCTTTGCCATCCTCCTG 79 CTGTCTCTCCTGCACTGA 80GGTGCAGCCTCACTGTCT 81 AACTGCCTGTTTGCCCAC 82 CTTCTGCCTGCACCCCTG 83ACTGACTGGGCATAGCTC 84

[0283] The sequences shown in Table 6-a above and Table 6-B below are ina 5′ to 3′ direction. This is reversed with respect to 3′ to 5′direction shown in the seq files of Example 3. For synthesis purposes,the seq files are generated reading from 3′ to 5′. This allows foraligning all of the 3′ most ‘A’ nucleosides together, all of the 3′ most‘G’ nucleosides together, all of the 3′ most ° C.′ nucleosides togetherand all of the 3′ most ‘T’ nucleosides together. Thus when the firstnucleoside of each particular oligonucleotide (attached to the solidsupport) is added to the wells on the plates, machine movement isreduced since an automatic pipette can move in a linear manner down onerow and up another on the 96 well plate.

[0284] The location of the well holding each particular oligonucleotidesis indicated by row and column. There are eight rows designated A to Gand twelve columns designated 1 to 12 in a typical 96 well format plate.Any particular well location is indicated by its ‘Well No.’ which isindicated by the combination of the row and the column, e.g. A08 is thewell at row A, column 8.

[0285] In Table 6-b below, the oligonucleotide of Table 6-a are shownreordered according to the Well No. on their synthesis plate. The ordershown in Table 6-b is the actually order as synthesized on an automatedsynthesizer taking advantage of the preferred placement of the firstnucleoside according to the above alignment criteria. TABLE 6-bSequences of Oligonucleotides Targeted to CD40 Order by Synthesis WellNo. Well SEQ ID A01 GACCAGGCGGCAGGACCA 2 A02 AGGTGAGACCAGGCGGCA 3 A03GCAGAGGCAGACGAACCA 5 A04 GCAAGCAGCCCCAGAGGA 6 A05 GGTCAGCAAGCAGCCCCA 7A06 GACAGCGGTCAGCAAGCA 8 A07 GATGGACAGCGGTCAGCA 9 A08 GGTGGTTCTGGATGGACA11 A09 GCAGTGGGTGGTTCTGGA 13 A10 CACAAAGAACAGCACTGA 14 A11CTGGCACAAAGAACAGCA 15 A12 TCCTGGCTGGCACAAAGA 16 B01 CTGTCCTGGCTGGCAcAA17 B02 ACTCTGTGCAGTCACTCA 21 B03 TTCACCGCAAGGAAGGCA 25 B04CTCTGTTCCAGGTGTCTA 26 B05 GTGCCAGCCTTCTTCACA 37 B06 TGCAGGACACAGCTCTCA40 B07 AATCTGCTTGACCCCAAA 43 B08 GTATCAGAAACCCCTGTA 45 B09GACTGGGCAGGGCTCGCA 49 B10 CATTGGAGAAGAAGCCGA 50 B11 TCGAAAGCAGATGACACA53 B12 CAGGTCTTTGGTCTCACA 57 C01 TTTTGATAAAGACCAGCA 68 C02GATCGTCGGGAAAATTGA 72 C03 TGGAGCAGCAGTGTTGGA 75 C04 CGGTTGGCATCCATGTAA78 C05 CTGTCTCTCCTGCACTGA 80 C06 TCTGGATGGACAGCGGTC 10 C07CTGGTGGCAGTGTGTCTC 27 C08 GGTGCCCTTCTGCTGGAC 31 C09 ACAGGTGCAGATGGTGTC35 C10 GAAACCCCTGTAGCAATC 44 C11 TTGGTCTCACAGCTTGTC 56 C12CTGTTGCACAACCAGGTC 58 D01 GTCTTGTTTGTGCCTGCC 60 D02 CCACAGACAACATCAGTC61 D03 CTGGGGACCACAGACAAC 62 D04 TCAGCCGATCCTGGGGAC 63 D05GTCGGGAAAATTGATCTC 71 D06 GGAGCCAGGAAGATCGTC 73 D07 TGGCATCCATGTAAAGTC77 D08 AACTGCCTGTTTGCCCAC 82 D09 ACTGACTGGGCATAGCTC 84 D10GTGGGTGGTTCTGGATGG 12 D11 GAAGGCATTCCGTTTCAG 24 D12 GTGTCTGTTTCTGAGGTG33 E01 TGGTGTCTGTTTCTGAGG 34 E02 GGACACAGCTCTCACAGG 39 E03GAGCGGTGCAGGACACAG 41 E04 AAGCCGGGCGAGCATGAG 42 E05 GCTCGCAGATGGTATCAG46 E06 GATGACACATTGGAGAAG 51 E07 GCAGATGACACATTGGAG 52 E08GTTTGTGCCTGCCTGTTG 59 E09 CACCACCAGGGCTCTCAG 64 E10 ACCAGCACCAAGAGGATG67 E11 TATTGGTTGGCTTCTTGG 69 E12 GGGTTCCTGCTTGGGGTG 70 F01GTAAAGTCTCCTGCACTG 76 F02 CTCTTTGCCATCCTCCTG 79 F03 CTTCTGCCTGCACCCCTG83 F04 CCAGGCGGCAGGACCACT 1 F05 CAGAGGCAGACGAACCAT 4 F06CTCACCAGTTTCTGTCCT 18 F07 TCACTCACCAGTTTCTGT 19 F08 GTGCAGTCACTCACCAGT20 F09 CAGTGAACTCTGTGCAGT 22 F10 ATTCCGTTTCAGTGAACT 23 F11TGGGGTCGCAGTATTTGT 28 F12 GGTTGGGGTCGCAGTATT 29 G01 CTAGGTTGGGGTCGCAGT30 G02 CTGAGGTGCCCTTCTGCT 32 G03 TTCACAGGTGCAGATGGT 36 G04TACAGTGCCAGCCTTCTT 38 G05 GCAGGGCTCGCAGATGGT 47 G06 TGGGCAGGGCTCGCAGAT48 G07 GTCCAAGGGTGACATTTT 54 G08 CACAGCTTGTCCAAGGGT 55 G09GGGATCACCACCAGGGCT 65 G10 GAGGATGGCAAACAGGAT 66 G11 TGGAGCCAGGAAGATCGT74 G12 GGTGCAGCCTCACTGTCT 81

Example 9 Oligonucleotide Analysis

[0286] A. Oligonucleotide Analysis—96 Well Plate Format

[0287] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman MDQ) or, forindividually prepared samples, on a commercial CE apparatus (e.g.,Beckman 5000, ABI 270). Base and backbone composition was confirmed bymass analysis of the compounds utilizing electrospray-mass spectroscopy.All assay test plates were diluted from the master plate using singleand multi-channel robotic pipettors.

[0288] B. Alternative Oligonucleotide Analysis

[0289] After cleavage from the controlled pore glass support (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides are analyzed by polyacrylamide gelelectrophoresis on denaturing gels. Oligonucleotide purity is checked by³¹P nuclear magnetic resonance spectroscopy, and/or by HPLC, asdescribed by Chiang et al., J. Biol. Chem. 1991, 266, 18162.

Example 10 Automated Assay of CD40 Oligonucleotides

[0290] A. Poly(A)+ mRNA Isolation.

[0291] Poly(A)+ mRNA was isolated according to Miura et al. (Clin.Chem., 1996, 42, 1758). Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μl cold PBS. 60 μl lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA,0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was addedto each well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μl of lysate was transferred to Oligod(T) coated 96 well plates (AGCT Inc., Irvine, Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μlof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μl of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C. was added to each well, the platewas incubated on a 90° C. plate for 5 minutes, and the eluate thentransferred to a fresh 96-well plate. Cells grown on 100 mm or otherstandard plates may be treated similarly, using appropriate volumes ofall solutions.

[0292] B. RT-PCR Analysis of CD40 mRNA Levels

[0293] Quantitation of CD40 mRNA levels was determined by reversetranscriptase polymerase chain reaction (RT-PCR) using the ABIPRISM™_(—)7700 Sequence Detection System (PE-Applied Biosystems, FosterCity, Calif.) according to manufacturer's instructions. This is aclosed-tube, non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time.

[0294] As opposed to standard PCR, in which amplification products arequantitated after the PCR is completed, products in RT-PCR arequantitated as they accumulate. This is accomplished by including in thePCR reaction an oligonucleotide probe that anneals specifically betweenthe forward and reverse PCR primers, and contains two fluorescent dyes.A reporter dye (e.g., JOE or FAM, PE-Applied Biosystems, Foster City,Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g.,TAMRA, PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated.

[0295] With each cycle, additional reporter dye molecules are cleavedfrom their respective probes, and the fluorescence intensity ismonitored at regular (six-second) intervals by laser optics built intothe ABI PRISM™_(—)7700 Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0296] RT-PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μl PCRcocktail (1× Taqman™ buffer A, 5.5 mM MgCl₂, 300 uM each of DATP, dCTPand dGTP, 600 uM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 U RNAse inhibitor, 1.25 units AmpliTaq Gold™, and 12.5 UMuLV reverse transcriptase) to 96 well plates containing 25 μl poly(A)mRNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. following a 10 minute incubation at 95° C. to activatethe AmpliTaq Gold™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0297] For CD40, the PCR primers were:

[0298] forward primer: forward primer: CAGAGTTCACTGAAACGGAATGC (SEQ IDNO:86) reverse primer: GGTGGCAGTGTGTCTCTCTGTTC (SEQ ID NO:87)

[0299] reverse primer:

[0300] and the PCR probe was: FAM-TTCCTTGCGGTGAAAGCGAATTCCT-TAMRA

[0301] (SEQ ID NO:88) where FAM (PE-Applied Biosystems, Foster City,Calif.) is the fluorescent reporter dye and TAMRA (PE-AppliedBiosystems, Foster City, Calif.) is the quencher dye.

[0302] For GAPDH the PCR primers were:

[0303] forward primer:

[0304] reverse primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:89)GAAGATGGTGATGGGATTTC, (SEQ ID NO:90)

[0305] and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQID No. 91) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 11 Inhibition of CD40 Expression by PhosphorothioateOligodeoxynucleotides

[0306] In accordance with the present invention, a series ofoligonucleotides complementary to mRNA were designed to target differentregions of the human CD40 mRNA, using published sequences (GenBankaccession number X60592, incorporated herein as SEQ ID NO: 85). Theoligonucleotides are shown in Table 7. Target sites are indicated by thebeginning nucleotide numbers, as given in the sequence source reference(X60592), to which the oligonucleotide binds. All compounds in Table 7are oligodeoxynucleotides with phosphorothioate backbones(internucleoside linkages) throughout. Data are averages from threeexperiments. TABLE 7 Inhibition of CD40 mRNA Levels by PhosphorothioateOligodeoxynucleotides TARGET SEQ ID ISIS# SITE SEQUENCE % INHIB. NO.18623 18 CCAGGCGGCAGGACCACT 30.71 1 18624 20 GACCAGGCGGCAGGACCA 28.09 218625 26 AGGTGAGACCAGGCGGCA 21.89 3 18626 48 CAGAGGCAGACGAACCAT 0.00 418627 49 GCAGAGGCAGACGAACCA 0.00 5 18628 73 GCAAGCAGCCCCAGAGGA 0.00 618629 78 GGTCAGCAAGCAGCCCCA 29.96 7 18630 84 GACAGCGGTCAGCAAGCA 0.00 818631 88 GATGGACAGCGGTCAGCA 0.00 9 18632 92 TCTGGATGGACAGCGGTC 0.00 1018633 98 GGTGGTTCTGGATGGACA 0.00 11 18634 101 GTGGGTGGTTCTGGATGG 0.00 1218635 104 GCAGTGGGTGGTTCTGGA 0.00 13 18636 152 CACAAAGAACAGCACTGA 0.0014 18637 156 CTGGCACAAAGAACAGCA 0.00 15 18638 162 TCCTGGCTGGCACAAAGA0.00 16 18639 165 CTGTCCTGGCTGGCACAA 4.99 17 18640 176CTCACCAGTTTCTGTCCT 0.00 18 18641 179 TCACTCACCAGTTTCTGT 0.00 19 18642185 GTGCAGTCACTCACCAGT 0.00 20 18643 190 ACTCTGTGCAGTCACTcA 0.00 2118644 196 CAGTGAACTCTGTGCAGT 5.30 22 18645 205 ATTCCGTTTCAGTGAACT 0.0023 18646 211 GAAGGCATTCCGTTTCAG 9.00 24 18647 222 TTCACCGCAAGGAAGGCA0.00 25 18648 250 CTCTGTTCCAGGTGTCTA 0.00 26 18649 267CTGGTGGCAGTGTGTCTC 0.00 27 18650 286 TGGGGTCGCAGTATTTGT 0.00 28 18651289 GGTTGGGGTCGCAGTATT 0.00 29 18652 292 CTAGGTTGGGGTCGCAGT 0.00 3018653 318 GGTGCCCTTCTGCTGGAC 19.67 31 18654 322 CTGAGGTGCCCTTCTGCT 15.6332 18655 332 GTGTCTGTTTCTGAGGTG 0.00 33 18656 334 TGGTGTCTGTTTCTGAGG0.00 34 18657 345 ACAGGTGCAGATGGTGTC 0.00 35 18658 348TTCACAGGTGCAGATGGT 0.00 36 18659 360 GTGCCAGCCTTCTTCACA 5.67 37 18660364 TACAGTGCCAGCCTTCTT 7.80 38 18661 391 GGACACAGCTCTCACAGG 0.00 3918662 395 TGCAGGACACAGCTCTCA 0.00 40 18663 401 GAGCGGTGCAGGACACAG 0.0041 18664 416 AAGCCGGGCGAGCATGAG 0.00 42 18665 432 AATCTGCTTGACCCCAAA5.59 43 18666 446 GAAACCCCTGTAGCAATC 0.10 44 18667 452GTATCAGAAACCCCTGTA 0.00 45 18668 463 GCTCGCAGATGGTATCAG 0.00 46 18669468 GCAGGGCTCGCAGATGGT 34.05 47 18670 471 TGGGCAGGGCTCGCAGAT 0.00 4818671 474 GACTGGGCAGGGCTCGCA 2.71 49 18672 490 CATTGGAGAAGAAGCCGA 0.0050 18673 497 GATGACACATTGGAGAAG 0.00 51 18674 500 GCAGATGACACATTGGAG0.00 52 18675 506 TCGAAAGCAGATGACACA 0.00 53 1.8676 524GTCCAAGGGTGACATTTT 8.01 54 18677 532 CACAGCTTGTCCAAGGGT 0.00 55 18678539 TTGGTCTCACAGCTTGTC 0.00 56 18679 546 CAGGTCTTTGGTCTCACA 6.98 5718680 558 CTGTTGCACAACCAGGTC 18.76 58 18681 570 GTTTGTGCCTGCCTGTTG 2.4359 18682 575 GTCTTGTTTGTGCCTGCC 0.00 60 18683 590 CCACAGACAACATCAGTC0.00 61 18684 597 CTGGGGACCACAGACAAC 0.00 62 18685 607TCAGCCGATCCTGGGGAC 0.00 63 18686 621 CACCACCAGGGCTCTCAG 23.31 64 18687626 GGGATCACCACCAGGGCT 0.00 65 18688 657 GAGGATGGCAAACAGGAT 0.00 6618689 668 ACCAGCACCAAGAGGATG 0.00 67 18690 679 TTTTGATAAAGACCAGCA 0.0068 18691 703 TATTGGTTGGCTTCTTGG 0.00 69 18692 729 GGGTTCCTGCTTGGGGTG0.00 70 18693 750 GTCGGGAAAATTGATCTC 0.00 71 18694 754GATCGTCGGGAAAATTGA 0.00 72 18695 765 GGAGCCAGGAAGATCGTC 0.00 73 18696766 TGGAGCCAGGAAGATCGT 0.00 74 18697 780 TGGAGCAGCAGTGTTGGA 0.00 7518698 796 GTAAAGTCTCCTGCACTG 0.00 76 18699 806 TGGCATCCATGTAAAGTC 0.0077 18700 810 CGGTTGGCATCCATGTAA 0.00 78 18701 834 CTCTTTGCCATCCTCCTG4.38 79 18702 861 CTGTCTCTCCTGCACTGA 0.00 80 18703 873GGTGCAGCCTCACTGTCT 0.00 81 18704 910 AACTGCCTGTTTGCCCAC 33.89 82 18705954 CTTCTGCCTGCACCCCTG 0.00 83 18706 976 ACTGACTGGGCATAGCTC 0.00 84

Example 12 Inhibition of CD40 Expression by Phosphoro-Thioate 2′-MOEGapmer Oligonucleotides

[0307] In accordance with the present invention, a second series ofoligonucleotides complementary to mRNA were designed to target differentregions of the human CD40 mRNA, using published sequence X60592. Theoligonucleotides are shown in Table 8. Target sites are indicated by thebeginning or initial nucleotide numbers, as given in the sequence sourcereference (X60592), to which the oligonucleotide binds.

[0308] All compounds in Table 8 are chimeric oligonucleotides(‘gapmers’) 18 nucleotides in length, composed of a central ‘gap’ regionconsisting of ten 2′deoxynucleotides, which is flanked on both sides (5′and 3′ directions) by four-nucleotide ‘wings.’ The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The intersugar (backbone) linkagesare phosphorothioate (P═S) throughout the oligonucleotide. Cytidineresidues in the 2′-MOE wings are 5-methylcytidines.

[0309] Data are averaged from three experiments. TABLE 8 Inhibition ofCD40 mRNA Levels by Chimeric Phosphorothioate Oligonucleotides TARGETSEQ ID ISIS# SITE SEQUENCE % Inhibition NO. 19211 18 CCAGGCGGCAGGACCACT75.71 1 19212 20 GACCAGGCGGCAGGACCA 77.23 2 19213 26 AGGTGAGACCAGGCGGCA80.82 3 19214 48 CAGAGGCAGACGAACCAT 23.68 4 19215 49 GCAGAGGCAGACGAACCA45.97 5 19216 73 GCAAGCAGCCCCAGAGGA 65.80 6 19217 78 GGTCAGCAAGCAGCCCCA74.73 7 19218 84 GACAGCGGTCAGCAAGCA 67.21 8 19219 88 GATGGACAGCGGTCAGCA65.14 9 19220 92 TCTGGATGGACAGCGGTC 78.71 10 19221 98 GGTGGTTCTGGATGGACA81.33 11 19222 101 GTGGGTGGTTCTGGATGG 57.79 12 19223 104GCAGTGGGTGGTTCTGGA 73.70 13 19224 152 CACAAAGAACAGCACTGA 40.25 14 19225156 CTGGCACAAAGAACAGCA 60.11 15 19226 162 TCCTGGCTGGCACAAAGA 10.18 1619227 165 CTGTCCTGGCTGGCACAA 24.37 17 19228 176 CTCACCAGTTTCTGTCCT 22.3018 19229 179 TCACTCACCAGTTTCTGT 40.64 19 19230 185 GTGCAGTCACTCACCAGT82.04 20 19231 190 ACTCTGTGCAGTCACTCA 37.59 21 19232 196CAGTGAACTCTGTGCAGT 40.26 22 19233 205 ATTCCGTTTCAGTGAACT 56.03 23 19234211 GAAGGCATTCCGTTTCAG 32.21 24 19235 222 TTCACCGCAAGGAAGGCA 61.03 2519236 250 CTCTGTTCCAGGTGTCTA 62.19 26 19237 267 CTGGTGGCAGTGTGTCTC 70.3227 19238 286 TGGGGTCGCAGTATTTGT 0.00 28 19239 289 GGTTGGGGTCGCAGTATT19.40 29 19240 292 CTAGGTTGGGGTCGCAGT 36.32 30 19241 318GGTGCCCTTCTGCTGGAC 78.91 31 19242 322 CTGAGGTGCCCTTCTGCT 69.84 32 19243332 GTGTCTGTTTCTGAGGTG 63.32 33 19244 334 TGGTGTCTGTTTCTGAGG 42.83 3419245 345 ACAGGTGCAGATGGTGTC 73.31 35 19246 348 TTCACAGGTGCAGATGGT 47.7236 19247 360 GTGCCAGCCTTCTTCACA 61.32 37 19248 364 TACAGTGCCAGCCTTCTT46.82 38 19249 391 GGACACAGCTCTCACAGG 0.00 39 19250 395TGCAGGACACAGCTCTCA 52.05 40 19251 401 GAGCGGTGCAGGACACAG 50.15 41 19252416 AAGCCGGGCGAGCATGAG 32.36 42 19253 432 AATCTGCTTGACCCCAAA 0.00 4319254 446 GAAACCCCTGTAGCAATC 0.00 44 19255 452 GTATCAGAAACCCCTGTA 36.1345 19256 463 GCTCGCAGATGGTATCAG 64.65 46 19257 468 GCAGGGCTCGCAGATGGT74.95 47 19258 471 TGGGCAGGGCTCGCAGAT 0.00 48 19259 474GACTGGGCAGGGCTCGCA 82.00 49 19260 490 CATTGGAGAAGAAGCCGA 41.31 50 19261497 GATGACACATTGGAGAAG 13.81 51 19262 500 GCAGATGACACATTGGAG 78.48 5219263 506 TCGAAAGCAGATGACACA 59.28 53 19264 524 GTCCAAGGGTGACATTTT 70.9954 19265 532 CACAGCTTGTCCAAGGGT 0.00 55 19266 539 TTGGTCTCACAGCTTGTC45.92 56 19267 546 CAGGTCTTTGGTCTCACA 63.95 57 19268 558CTGTTGCACAACCAGGTC 82.32 58 19269 570 GTTTGTGCCTGCCTGTTG 70.10 59 19270575 GTCTTGTTTGTGCCTGCC 68.95 60 19271 590 CCACAGACAACATCAGTC 11.22 6119272 597 CTGGGGACCACAGACAAC 9.04 62 19273 607 TCAGCCGATCCTGGGGAC 0.0063 19274 621 CACCACCAGGGCTCTCAG 23.08 64 19275 626 GGGATCACCACCAGGGCT57.94 65 19276 657 GAGGATGGCAAACAGGAT 49.14 66 19277 668ACCAGCACCAAGAGGATG 3.48 67 19278 679 TTTTGATAAAGACCAGCA 30.58 68 19279703 TATTGGTTGGCTTCTTGG 49.26 69 19280 729 GGGTTCCTGCTTGGGGTG 13.95 7019281 750 GTCGGGAAAATTGATCTC 54.78 71 19282 754 GATCGTCGGGAAAATTGA 0.0072 19283 765 GGAGCCAGGAAGATCGTC 69.47 73 19284 766 TGGAGCCAGGAAGATCGT54.48 74 19285 780 TGGAGCAGCAGTGTTGGA 15.17 75 19286 796GTAAAGTCTCCTGCACTG 30.62 76 19287 806 TGGCATCCATGTAAAGTC 65.03 77 19288810 CGGTTGGCATCCATGTAA 34.49 78 19289 834 CTCTTTGCCATCCTCCTG 41.84 7919290 861 CTGTCTCTCCTGCACTGA 25.68 80 19291 873 GGTGCAGCCTCACTGTCT 76.2781 19292 910 AACTGCCTGTTTGCCCAC 63.34 82 19293 954 CTTCTGCCTGCACCCCTG0.00 83 19294 976 ACTGACTGGGCATAGCTC 11.55 84

Example 13 Oligonucleotide-Sensitive Sites of the CD40 Target NucleicAcid

[0310] As the data presented in the preceding two Examples shows,several sequences were present in preferred compounds of two distinctoligonucleotide chemistries. Specifically, compounds having SEQ ID NOS:1, 2, 7, 47 and 82 are preferred in both instances. These compounds mapto different regions of the CD40 transcript but nevertheless defineaccessible sites of the target nucleic acid.

[0311] For example, SEQ ID NOS: 1 and 2 overlap each other and both mapto the 5-untranslated region (5′-UTR) of CD40. Accordingly, this regionof CD40 is particularly preferred for modulation via sequence-basedtechnologies. Similarly, SEQ ID NOS: 7 and 47 map to the open readingframe of CD40, whereas SEQ ID NO: 82 maps to the 3′-untranslated region(3′-UTR. Thus, the ORF and 3′-UTR of CD40 may be targeted bysequence-based technologies as well.

[0312] The reverse complements of the active CD40 compounds are easilydetermined by those skilled in the art and may be assembled to yieldnucleotide sequences corresponding to accessible sites on the targetnucleic acid. For example, the assembled reverse complement of SEQ IDNOS: 1 and 2 is represented below as SEQ ID NO:92: 5′-AGTGGTCCTGCCGCCTGGTC -3′ SEQ ID NO:92     ||||||||||||||||||||    TCACCAGGACGGCGGACC   -5′ SEQ ID NO:1       ACCAGGACGGCGGACCAG -5′SEQ ID NO:2

[0313] Through multiple iterations of the process of the invention, moreextensive ‘footprints’ are generated. A library of this information iscompiled and may be used by those skilled in the art in a variety ofsequence-based technologies to study the molecular and biologicalfunctions of CD40 and to investigate or confirm its role in variousdiseases and disorders.

Example 14 Site Selection Program

[0314] In a preferred embodiment of the invention, illustrated in FIG.20, an application is deployed which facilitates the selection processfor determining the target positions of the oligos to be synthesized, or‘sites.’ This program is written using a three-tiered object-orientedapproach. All aspects of the software described, therefore, are tightlyintegrated with the relational database. For this reason, explicitdatabase read and write steps are not shown. It should be assumed thateach step described includes database access. The description belowillustrates one way the program can be used. The actual interface allowsusers to skip from process to process at will, in any order.

[0315] Before running the site picking program, the target must have allrelevant properties computed as described previously and indicated inprocess step 2204. When the site picking program is launched at processstep 2206 the user is presented with a panel showing targets which havepreviously been selected and had their properties calculated. The userselects one target to work with at process step 2208 and proceeds todecide if any derived properties will be needed at process step 2210.Derived properties are calculated by performing mathematical operationson combinations of pre-calculated properties as defined by the user atprocess step 2212.

[0316] The derived properties are made available as peers with all thepre-calculated properties. The user selects one of the properties toview plotted versus target position at process step 2214. This graph isshown above a linear representation of the target. The horizontal orposition axis of both the graph and target are linked and scalable bythe user. The zoom range goes from showing the full target length toshowing individual target bases as letters and individual propertypoints. The user next selects a threshold value below or above which allsites will be eliminated from future consideration at process step 2216.The user decides whether to eliminate more sites based on any otherproperties at process step 2218. If they choose to eliminate more, theyreturn to pick another property to display at process step 2214 andthreshold at process step 2216.

[0317] After eliminating sites, the user selects from the remaining listby choosing any property at process step 2220 and then choosing a manualor automatic selection technique at process step 2222. In the automatictechnique, the user decides whether they want to pick from maxima orminima and the number of maxima or minima to be selected as sites atprocess step 2224. The software automatically finds and picks thepoints. When picking manually the user must decide if they wish to useautomatic peak finding at process step 2226. If the user selectsautomatic peak finding, then user must click on the graphed propertywith the mouse at process step 2236. The nearest maxima or minima,depending on the modifier key held down, to the selected point will bepicked as the site. Without the peak finding option, the user must picka site at process step 2238 by clicking on its position on the linearrepresentation of target.

[0318] Each time a site, or group of sites, is picked, a dynamicproperty is calculated for all possible sites (not yet eliminated) atprocess step 2230. This property indicates the nearness of the site twoa picked site allowing the user to pick sites in subsequent iterationsbased on target coverage. After new sites are picked, the userdetermines if the desired number of sites has been picked. If too fewsites have been picked the user returns to pick more 2220. If too manysites have been picked, the user may eliminate them by selecting anddeleting them on the target display at process step 2234. If the correctnumber of sites is picked, and the user is satisfied with he set ofpicked sites, the user registers these sites to the database along withtheir name, notebook number, and page number at process step 2238. Thedatabase time stamps this registration event.

Example 15 Site Selection Program

[0319] In a preferred embodiment of the invention, illustrated in FIG.21, an application is deployed which facilitates the assignment ofspecific chemical structure to the complement of the sequence of thesites previously picked and facilitates the registration and ordering ofthese now fully defined antisense compounds. This program is writtenusing a three-tiered object-oriented approach. All aspects of thesoftware described, therefore, are tightly integrated with therelational database. For this reason, explicit database read and writesteps are not shown, it being understood that each step described alsoincludes appropriate database read/write access.

[0320] To begin using the oligonucleotide chemistry assignment program,the user launches it at process step 2302. The user then selects fromthe previously selected sets of oligonucleotides at process step 2304,registered to the database in site picker's process step 2238. Next, theuser must decide whether to manually assign the chemistry a base at atime, or run the sites through a template at process step 2306. If theuser chooses to use a template, they must determine if a desiredtemplate is available at process step 2308. If a template is notavailable with the desired chemistry modifications and the correctlength, the user can define one at process step 2314.

[0321] To define a template, the user must select the length of theoligonucleotide the template is to define. This oligonucleotide is thenrepresented as a bar with selectable regions. The user sets the numberof regions on the oligonucleotide, and the positions and lengths ofthese regions by dragging them back and forth on the bar. Each region isrepresented by a different color.

[0322] For each region, the user defines the chemistry modifications forthe sugars, the linkers, and the heterocycles at each base position inthe region. At least four heterocycle chemistries must be given, one foreach of the four possible base types (A, G, C or T or U) in the sitesequence the template will be applied to. A user interface is providedto select these chemistries which show the molecular structure of eachcomponent selected and its modification name. By pushing on a pop-uplist next to each of the pictures, the user may choose from a list ofstructures and names, those possible to put in this place. For example,the heterocycle that represents the base type G is shown as a twodimensional structure diagram. If the user clicks on the pop-up list, arow of other possible structures and names is shown. The user drags themouse to the desired chemistry and releases the mouse. Now the newlyselected molecule is displayed as the choice for G type heterocyclemodifications.

[0323] Once the user has created a template, or selected an existingone, the software applies the template at process step 2312 to each ofthe complements of the sites in the list. When the templates areapplied, it is possible that chemistries will be defined which areimpossible to make with the chemical precursors presently used on theautomatic synthesizer. To check this, a database is maintained of allprecursors previously designed, and their availability for automatedsynthesis. When the templates are applied, the resulting molecules aretested at process step 2316 against this database to see if they arereadily synthesized.

[0324] If a molecule is not readily synthesized, it is added to a listthat the user inspects. At process step 2318, the user decides whetherto modify the chemistry to make it compatible with the currentlyrecognized list of available chemistries or to ignore it. To modify achemistry, the user must use the base at a time interface at processstep 2322. The user can also choose to go directly to this step,bypassing templates all together at process step 2306.

[0325] The base at a time interface at process step 2322 is very similarto the template editor at process step 2314 except that instead ofspecifying chemistries for regions, they are defined one base at a time.This interface also differs in that it dynamically checks to see if thedesign is readily synthesized as the user makes selections. In otherwords, each choice made limits the choices the software makes availableon the pop-up selection lists. To accommodate this function, anadditional choice is made available on each pop-up of ‘not defined.’ Forexample, this allows the user to inhibit linker choice from restrictingthe sugar choices by first setting the linker to ‘not defined.’ The userwould then pick the sugar, and then pick from the remaining linkerchoices available.

[0326] Once all of the sites on the list are assigned chemistries ordropped, they are registered at process step 2324 to the commercialchemical structure database. Registering to this database makes sure thestructure is unique, assigns it a new identifier if it is unique, andallows future structure and substructure searching by creating varioushash-tables. The compound definition is also stored at process step 2326to various hash tables referred to as chemistry/position tables. Theseallow antisense compound searching and categorization based onoligonucleotide chemistry modification sequences and equivalent basesequences.

[0327] The results of the registration are displayed at process step2328 with the new IDs if they are new compounds and with the old IDs ifthey have been previously registered. The user next selects which of thecompounds processed they wish to order for synthesis at process step2330 and registers an order list at process step 2332 by includingscientist name, notebook number and page number. The databasetime-stamps this entry. The user may than choose at process step 2334 toquit the program at process step 2338, go back to the beginning andchoose a new site list to work with process step 2304, or start theoligonucleotide ordering interface at process step 2336.

Example 16 Gene Walk to Optimize Oligonucleotide Sequence

[0328] A gene walk is executed using a CD40 antisense oligonucleotidehaving SEQ ID NO:15 (5′-CTGGCACAAAGAACAGCA. In effecting this gene walk,the following parameters are used: Gene Walk Parameter Entered valueOligonucleotide Sequence ID: 15 Name of Gene Target: CD40 Scope of GeneWalk: 20 Sequence Shift Increment:  1

[0329] Entering these values and effecting the gene walk centered on SEQID NO: 15 automatically generates the following new oligonucleotides:TABLE 8 Oligonucleotide Generated By Gene Walk SEQ ID Sequence 93GAACAGCACTGACTGTTT 94 AGAACAGCACTGACTGTT 95 AAGAACAGCACTGACTGT 96AAAGAACAGCACTGACTG 97 CAAAGAACAGCACTGACT 98 ACAAAGAACAGCACTGAC 99CACAAAGAACAGCACTGA 100 GCACAAAGAACAGCACTG 101 GGCACAAAGAACAGCACT 102TGGCACAAAGAACAGCAC 15 CTGGCACAAAGAACAGCA 103 GCTGGCACAAAGAACAGC 104GGCTGGCACAAAGAACAG 105 TGGCTGGCACAAAGAACA 106 CTGGCTGGCACAAAGAAC 107CCTGGCTGGCACAAAGAA 108 TCCTGGCTGGCACAAAGA 109 GTCCTGGCTGGCACAAAG 110TGTCCTGGCTGGCACAAA 111 CTGTCCTGGCTGGCACAA 112 TCTGTCCTGGCTGGCACA

[0330] The list shown above contains 20 oligonucleotide sequencesdirected against the CD40 nucleic acid sequence. They are ordered by theposition along the CD40 sequence at which the 5 ′ terminus of eacholigonucleotide hybridizes. This, the first ten oligonucleotides aresingle-base frame shift sequences directed against the CD40 sequenceupstream of compound SEQ ID NO: 15 and the latter ten are single-baseframe shift sequences directed against the CD40 sequence downstream ofcompound SEQ ID NO: 15.

1 112 1 18 DNA Artificial Sequence Novel Sequence 1 ccaggcggca ggaccact18 2 18 DNA Artificial Sequence Novel Sequence 2 gaccaggcgg caggacca 183 18 DNA Artificial Sequence Novel Sequence 3 aggtgagacc aggcggca 18 418 DNA Artificial Sequence Novel Sequence 4 cagaggcaga cgaaccat 18 5 18DNA Artificial Sequence Novel Sequence 5 gcagaggcag acgaacca 18 6 18 DNAArtificial Sequence Novel Sequence 6 gcaagcagcc ccagagga 18 7 18 DNAArtificial Sequence Novel Sequence 7 ggtcagcaag cagcccca 18 8 18 DNAArtificial Sequence Novel Sequence 8 gacagcggtc agcaagca 18 9 18 DNAArtificial Sequence Novel Sequence 9 gatggacagc ggtcagca 18 10 18 DNAArtificial Sequence Novel Sequence 10 tctggatgga cagcggtc 18 11 18 DNAArtificial Sequence Novel Sequence 11 ggtggttctg gatggaca 18 12 18 DNAArtificial Sequence Novel Sequence 12 gtgggtggtt ctggatgg 18 13 18 DNAArtificial Sequence Novel Sequence 13 gcagtgggtg gttctgga 18 14 18 DNAArtificial Sequence Novel Sequence 14 cacaaagaac agcactga 18 15 18 DNAArtificial Sequence Novel Sequence 15 ctggcacaaa gaacagca 18 16 18 DNAArtificial Sequence Novel Sequence 16 tcctggctgg cacaaaga 18 17 18 DNAArtificial Sequence Novel Sequence 17 ctgtcctggc tggcacaa 18 18 18 DNAArtificial Sequence Novel Sequence 18 ctcaccagtt tctgtcct 18 19 18 DNAArtificial Sequence Novel Sequence 19 tcactcacca gtttctgt 18 20 18 DNAArtificial Sequence Novel Sequence 20 gtgcagtcac tcaccagt 18 21 18 DNAArtificial Sequence Novel Sequence 21 actctgtgca gtcactca 18 22 18 DNAArtificial Sequence Novel Sequence 22 cagtgaactc tgtgcagt 18 23 18 DNAArtificial Sequence Novel Sequence 23 attccgtttc agtgaact 18 24 18 DNAArtificial Sequence Novel Sequence 24 gaaggcattc cgtttcag 18 25 18 DNAArtificial Sequence Novel Sequence 25 ttcaccgcaa ggaaggca 18 26 18 DNAArtificial Sequence Novel Sequence 26 ctctgttcca ggtgtcta 18 27 18 DNAArtificial Sequence Novel Sequence 27 ctggtggcag tgtgtctc 18 28 18 DNAArtificial Sequence Novel Sequence 28 tggggtcgca gtatttgt 18 29 18 DNAArtificial Sequence Novel Sequence 29 ggttggggtc gcagtatt 18 30 18 DNAArtificial Sequence Novel Sequence 30 ctaggttggg gtcgcagt 18 31 18 DNAArtificial Sequence Novel Sequence 31 ggtgcccttc tgctggac 18 32 18 DNAArtificial Sequence Novel Sequence 32 ctgaggtgcc cttctgct 18 33 18 DNAArtificial Sequence Novel Sequence 33 gtgtctgttt ctgaggtg 18 34 18 DNAArtificial Sequence Novel Sequence 34 tggtgtctgt ttctgagg 18 35 18 DNAArtificial Sequence Novel Sequence 35 acaggtgcag atggtgtc 18 36 18 DNAArtificial Sequence Novel Sequence 36 ttcacaggtg cagatggt 18 37 18 DNAArtificial Sequence Novel Sequence 37 gtgccagcct tcttcaca 18 38 18 DNAArtificial Sequence Novel Sequence 38 tacagtgcca gccttctt 18 39 18 DNAArtificial Sequence Novel Sequence 39 ggacacagct ctcacagg 18 40 18 DNAArtificial Sequence Novel Sequence 40 tgcaggacac agctctca 18 41 18 DNAArtificial Sequence Novel Sequence 41 gagcggtgca ggacacag 18 42 18 DNAArtificial Sequence Novel Sequence 42 aagccgggcg agcatgag 18 43 18 DNAArtificial Sequence Novel Sequence 43 aatctgcttg accccaaa 18 44 18 DNAArtificial Sequence Novel Sequence 44 gaaacccctg tagcaatc 18 45 18 DNAArtificial Sequence Novel Sequence 45 gtatcagaaa cccctgta 18 46 18 DNAArtificial Sequence Novel Sequence 46 gctcgcagat ggtatcag 18 47 18 DNAArtificial Sequence Novel Sequence 47 gcagggctcg cagatggt 18 48 18 DNAArtificial Sequence Novel Sequence 48 tgggcagggc tcgcagat 18 49 18 DNAArtificial Sequence Novel Sequence 49 gactgggcag ggctcgca 18 50 18 DNAArtificial Sequence Novel Sequence 50 cattggagaa gaagccga 18 51 18 DNAArtificial Sequence Novel Sequence 51 gatgacacat tggagaag 18 52 18 DNAArtificial Sequence Novel Sequence 52 gcagatgaca cattggag 18 53 18 DNAArtificial Sequence Novel Sequence 53 tcgaaagcag atgacaca 18 54 18 DNAArtificial Sequence Novel Sequence 54 gtccaagggt gacatttt 18 55 18 DNAArtificial Sequence Novel Sequence 55 cacagcttgt ccaagggt 18 56 18 DNAArtificial Sequence Novel Sequence 56 ttggtctcac agcttgtc 18 57 18 DNAArtificial Sequence Novel Sequence 57 caggtctttg gtctcaca 18 58 18 DNAArtificial Sequence Novel Sequence 58 ctgttgcaca accaggtc 18 59 18 DNAArtificial Sequence Novel Sequence 59 gtttgtgcct gcctgttg 18 60 18 DNAArtificial Sequence Novel Sequence 60 gtcttgtttg tgcctgcc 18 61 18 DNAArtificial Sequence Novel Sequence 61 ccacagacaa catcagtc 18 62 18 DNAArtificial Sequence Novel Sequence 62 ctggggacca cagacaac 18 63 18 DNAArtificial Sequence Novel Sequence 63 tcagccgatc ctggggac 18 64 18 DNAArtificial Sequence Novel Sequence 64 caccaccagg gctctcag 18 65 18 DNAArtificial Sequence Novel Sequence 65 gggatcacca ccagggct 18 66 18 DNAArtificial Sequence Novel Sequence 66 gaggatggca aacaggat 18 67 18 DNAArtificial Sequence Novel Sequence 67 accagcacca agaggatg 18 68 18 DNAArtificial Sequence Novel Sequence 68 ttttgataaa gaccagca 18 69 18 DNAArtificial Sequence Novel Sequence 69 tattggttgg cttcttgg 18 70 18 DNAArtificial Sequence Novel Sequence 70 gggttcctgc ttggggtg 18 71 18 DNAArtificial Sequence Novel Sequence 71 gtcgggaaaa ttgatctc 18 72 18 DNAArtificial Sequence Novel Sequence 72 gatcgtcggg aaaattga 18 73 18 DNAArtificial Sequence Novel Sequence 73 ggagccagga agatcgtc 18 74 18 DNAArtificial Sequence Novel Sequence 74 tggagccagg aagatcgt 18 75 18 DNAArtificial Sequence Novel Sequence 75 tggagcagca gtgttgga 18 76 18 DNAArtificial Sequence Novel Sequence 76 gtaaagtctc ctgcactg 18 77 18 DNAArtificial Sequence Novel Sequence 77 tggcatccat gtaaagtc 18 78 18 DNAArtificial Sequence Novel Sequence 78 cggttggcat ccatgtaa 18 79 18 DNAArtificial Sequence Novel Sequence 79 ctctttgcca tcctcctg 18 80 18 DNAArtificial Sequence Novel Sequence 80 ctgtctctcc tgcactga 18 81 18 DNAArtificial Sequence Novel Sequence 81 ggtgcagcct cactgtct 18 82 18 DNAArtificial Sequence Novel Sequence 82 aactgcctgt ttgcccac 18 83 18 DNAArtificial Sequence Novel Sequence 83 cttctgcctg cacccctg 18 84 18 DNAArtificial Sequence Novel Sequence 84 actgactggg catagctc 18 85 1004 DNAArtificial Sequence Novel Sequence 85 gcctcgctcg ggcgcccagt ggtcctgccgcctggtctca cctcgccatg gttcgtctgc 60 ctctgcagtg cgtcctctgg ggctgcttgctgaccgctgt ccatccagaa ccacccactg 120 catgcagaga aaaacagtac ctaataaacagtcagtgctg ttctttgtgc cagccaggac 180 agaaactggt gagtgactgc acagagttcactgaaacgga atgccttcct tgcggtgaaa 240 gcgaattcct agacacctgg aacagagagacacactgcca ccagcacaaa tactgcgacc 300 ccaacctagg gcttcgggtc cagcagaagggcacctcaga aacagacacc atctgcacct 360 gtgaagaagg ctggcactgt acgagtgaggcctgtgagag ctgtgtcctg caccgctcat 420 gctcgcccgg ctttggggtc aagcagattgctacaggggt ttctgatacc atctgcgagc 480 cctgcccagt cggcttcttc tccaatgtgtcatctgcttt cgaaaaatgt cacccttgga 540 caagctgtga gaccaaagac ctggttgtgcaacaggcagg cacaaacaag actgatgttg 600 tctgtggtcc ccaggatcgg ctgagagccctggtggtgat ccccatcatc ttcgggatcc 660 tgtttgccat cctcttggtg ctggtctttatcaaaaaggt ggccaagaag ccaaccaata 720 aggcccccca ccccaagcag gaaccccaggagatcaattt tcccgacgat cttcctggct 780 ccaacactgc tgctccagtg caggagactttacatggatg ccaaccggtc acccaggagg 840 atggcaaaga gagtcgcatc tcagtgcaggagagacagtg aggctgcacc cacccaggag 900 tgtggccacg tgggcaaaca ggcagttggccagagagcct ggtgctgctg ctgcaggggt 960 gcaggcagaa gcggggagct atgcccagtcagtgccagcc cctc 1004 86 23 DNA Artificial Sequence Novel Sequence 86cagagttcac tgaaacggaa tgc 23 87 23 DNA Artificial Sequence NovelSequence 87 ggtggcagtg tgtctctctg ttc 23 88 25 DNA Artificial SequenceNovel Sequence 88 ttccttgcgg tgaaagcgaa ttcct 25 89 19 DNA ArtificialSequence Novel Sequence 89 gaaggtgaag gtcggagtc 19 90 20 DNA ArtificialSequence Novel Sequence 90 gaagatggtg atgggatttc 20 91 20 DNA ArtificialSequence Novel Sequence 91 caagcttccc gttctcagcc 20 92 20 DNA ArtificialSequence Novel Sequence 92 agtggtcctg ccgcctggtc 20 93 18 DNA ArtificialSequence Novel Sequence 93 gaacagcact gactgttt 18 94 18 DNA ArtificialSequence Novel Sequence 94 agaacagcac tgactgtt 18 95 18 DNA ArtificialSequence Novel Sequence 95 aagaacagca ctgactgt 18 96 18 DNA ArtificialSequence Novel Sequence 96 aaagaacagc actgactg 18 97 18 DNA ArtificialSequence Novel Sequence 97 caaagaacag cactgact 18 98 18 DNA ArtificialSequence Novel Sequence 98 acaaagaaca gcactgac 18 99 18 DNA ArtificialSequence Novel Sequence 99 cacaaagaac agcactga 18 100 18 DNA ArtificialSequence Novel Sequence 100 gcacaaagaa cagcactg 18 101 18 DNA ArtificialSequence Novel Sequence 101 ggcacaaaga acagcact 18 102 18 DNA ArtificialSequence Novel Sequence 102 tggcacaaag aacagcac 18 103 18 DNA ArtificialSequence Novel Sequence 103 gctggcacaa agaacagc 18 104 18 DNA ArtificialSequence Novel Sequence 104 ggctggcaca aagaacag 18 105 18 DNA ArtificialSequence Novel Sequence 105 tggctggcac aaagaaca 18 106 18 DNA ArtificialSequence Novel Sequence 106 ctggctggca caaagaac 18 107 18 DNA ArtificialSequence Novel Sequence 107 cctggctggc acaaagaa 18 108 18 DNA ArtificialSequence Novel Sequence 108 tcctggctgg cacaaaga 18 109 18 DNA ArtificialSequence Novel Sequence 109 gtcctggctg gcacaaag 18 110 18 DNA ArtificialSequence Novel Sequence 110 tgtcctggct ggcacaaa 18 111 18 DNA ArtificialSequence Novel Sequence 111 ctgtcctggc tggcacaa 18 112 18 DNA ArtificialSequence Novel Sequence 112 tctgtcctgg ctggcaca 18

What is claimed is:
 1. A method of generating a set of compounds thatmodulate the expression of a target nucleic acid sequence comprisinggenerating a library of nucleobase sequences in silico according todefined criteria.
 2. A method of generating a set of compounds thatmodulate the expression of a target nucleic acid sequence comprisingevaluating in silico a plurality of virtual oligonucleotides accordingto defined criteria.
 3. A method of generating a set of compounds thatmodulate the expression of a target nucleic acid sequence comprisingrobotically synthesizing a plurality of defined oligonucleotidecompounds.
 4. A method of generating a set of compounds that modulatethe expression of a target nucleic acid sequence comprising roboticallyassaying a plurality of oligonucleotide compounds for one or moredesired physical, chemical or biological properties.
 5. A method ofgenerating a set of compounds that modulate the expression of a targetnucleic acid sequence comprising generating a library of nucleobasesequences in silico according to defined criteria and evaluating insilico a plurality of virtual oligonucleotides having said nucleobasesequences according to defined criteria.
 6. A method of generating a setof compounds that modulate the expression of a target nucleic acidsequence comprising evaluating in silico a plurality of virtualoligonucleotides according to defined criteria and roboticallysynthesizing a plurality of oligonucleotide compounds corresponding tosaid plurality of virtual oligonucleodites.
 7. A method of generating aset of compounds that modulate the expression of a target nucleic acidsequence comprising evaluating in silico a plurality of virtualoligonucleotides according to defined criteria and robotically assayinga plurality of oligonucleotide compounds corresponding to said virtualoligonucleotides for one or more desired physical, chemical orbiological properties.
 8. A method of generating a set of compounds thatmodulate the expression of a target nucleic acid sequence comprisinggenerating a library of nucleobase sequences in silico according todefined criteria and robotically synthesizing a plurality ofoligonucleotide compounds having said nucleobase sequences.
 9. A methodof generating a set of compounds that modulate the expression of atarget nucleic acid sequence comprising robotically synthesizing aplurality of oligonucleotide compounds and robotically assaying saidplurality of oligonucleotide compounds for one or more desired physical,chemical or biological properties.
 10. A method of generating a set ofcompounds that modulate the expression of a target nucleic acid sequencecomprising generating a library of nucleobase sequences in silicoaccording to defined criteria and robotically assaying a plurality ofoligonucleotide compounds having said nucleobase sequences for one ormore desired physical, chemical or biological properties.
 11. A methodof generating a set of compounds that modulate the expression of atarget nucleic acid sequence, comprising the steps of: (a) generating alibrary of nucleobase sequences in silico according to defined criteria;(b) evaluating in silico a plurality of virtual oligonucleotides havingthe nucleobase sequences of (a) according to defined criteria; and (c)robotically synthesizing a plurality of oligonucleotide compounds.
 12. Amethod of generating a set of compounds that modulate the expression ofa target nucleic acid sequence, comprising the steps of: (a) generatinga library of nucleobase sequences in silico according to definedcriteria; (b) evaluating in silico a plurality of virtualoligonucleotides having the nucleobase sequences of (a) according todefined criteria; and (c) robotically assaying a plurality ofoligonucleotide compounds for one or more desired physical, chemical orbiological properties.
 13. A method of generating a set of compoundsthat modulate the expression of a target nucleic acid sequence,comprising the steps of: (a) generating a library of nucleobasesequences in silico according to defined criteria; (b) roboticallysynthesizing a plurality of oligonucleotide compounds; and (c)robotically assaying a plurality of oligonucleotide compounds for one ormore desired physical, chemical or biological properties.
 14. A methodof generating a set of compounds that modulate the expression of atarget nucleic acid sequence, comprising the steps of: (a) evaluating insilico a plurality of virtual oligonucleotides according to definedcriteria; (b) robotically synthesizing a plurality of oligonucleotidecompounds; and (c) robotically assaying a plurality of oligonucleotidecompounds for one or more desired physical, chemical or biologicalproperties.
 15. A method of generating a set of compounds that modulatethe expression of a target nucleic acid sequence, comprising the stepsof: (a) generating a library of nucleobase sequences in silico accordingto defined criteria; (b) evaluating in silico a plurality of virtualoligonucleotides having the nucleobase sequences of (a) according todefined criteria; (c) robotically synthesizing a plurality ofoligonucleotide compounds; and (d) robotically assaying a plurality ofoligonucleotide compounds for one or more desired physical, chemical orbiological properties.
 16. A method of generating a set of compoundsthat modulate the expression of a target nucleic acid sequence,comprising the steps of: (a) generating a library of nucleobasesequences in silico according to defined criteria; (b) choosing anoligonucleotide chemistry; (c) robotically synthesizing a set ofoligonucleotide compounds having said nucleobase sequences of step (a)and said oligonucleotide chemistry of step (b); (d) robotically assayingsaid set of oligonucleotide compounds of step (c) for a physical,chemical or biological activity; and (e) selecting a subset of said setof oligonucleotide compounds of step (c) having a desired level ofphysical, chemical or biological activity in order to generate said setof compounds.
 17. A method of generating a set of compounds thatmodulate the expression of a target nucleic acid sequence, comprisingthe steps of: (a) generating a library of nucleobase sequences in silicoaccording to defined criteria; (b) choosing an oligonucleotidechemistry; (c) evaluating in silico a plurality of virtualoligonucleotides having the nucleobase sequences of (a) and theoligonucleotide chemistry of (b) according to defined criteria, andselecting those having preferred characteristics, in order to generate aset of preferred nucleobase sequences; (d) robotically synthesizing aset of oligonucleotide compounds having said preferred nucleobasesequences of step (c) and said oligonucleotide chemistry of step (b);(e) robotically assaying said set of oligonucleotide compounds of step(d) for a physical, chemical or biological activity; and (f) selecting asubset of said set of oligonucleotide compounds of step (d) having adesired level of physical, chemical or biological activity in order togenerate said set of compounds.
 18. The method of claim 4, wherein saidstep of robotically assaying said plurality of oligonucleotide compoundsis performed by computer-controlled real-time polymerase chain reactionor by computer-controlled enzyme-linked immunosorbent assay.
 19. Themethod of claim 1, wherein said target nucleic acid sequence is that ofa genomic DNA, a cDNA, a product of a polymerase chain reaction, anexpressed sequence tag, an mRNA or a structural RNA.
 20. The method ofclaim 1, wherein said target nucleic acid sequence is a human nucleicacid.
 21. A method of identifying one or more nucleic acid sequencesamenable to antisense modulation comprising generating a library ofantisense nucleobase sequences in silico according to defined criteria.22. A method of generating a set of compounds that modulate theexpression of a target nucleic acid sequence comprising evaluating insilico a plurality of virtual oligonucleotides according to definedcriteria.
 23. A method of identifying one or more nucleic acid sequencesamenable to antisense modulation comprising robotically synthesizing aplurality of antisense compounds.
 24. A method of identifying one ormore nucleic acid sequences amenable to antisense modulation comprisingrobotically assaying a plurality of antisense compounds for one or moredesired physical, chemical or biological properties.
 25. A method ofidentifying one or more nucleic acid sequences amenable to antisensemodulation comprising generating a library of nucleobase sequences insilico according to defined criteria and evaluating in silico aplurality of virtual oligonucleotides having said nucleobase sequencesaccording to defined criteria.
 26. A method of identifying one or morenucleic acid sequences amenable to antisense modulation comprisingevaluating in silico a plurality of virtual oligonucleotides accordingto defined criteria and robotically synthesizing a plurality ofoligonucleotide compounds.
 27. A method of identifying one or morenucleic acid sequences amenable to antisense modulation comprisingevaluating in silico a plurality of virtual oligonucleotides accordingto defined criteria and robotically assaying a plurality ofoligonucleotide compounds for one or more desired physical, chemical orbiological properties.
 28. A method of identifying one or more nucleicacid sequences amenable to antisense modulation comprising generating alibrary of nucleobase sequences in silico according to defined criteriaand robotically synthesizing a plurality of oligonucleotide compoundshaving said nucleobase sequences.
 29. A method of identifying one ormore nucleic acid sequences amenable to antisense modulation comprisingrobotically synthesizing a plurality of oligonucleotide compounds androbotically assaying said plurality of oligonucleotide compounds for oneor more desired physical, chemical or biological properties.
 30. Amethod of identifying one or more nucleic acid sequences amenable toantisense modulation comprising generating a library of nucleobasesequences in silico according to defined criteria and roboticallyassaying a plurality of oligonucleotide compounds having said nucleobasesequences for one or more desired physical, chemical or biologicalproperties.
 31. A method of identifying one or more nucleic acidsequences amenable to antisense modulation comprising the steps of: (a)generating a library of nucleobase sequences in silico according todefined criteria; (b) evaluating in silico a plurality of virtualoligonucleotides having the nucleobase sequences of (a) according todefined criteria; and (c) robotically synthesizing a plurality ofoligonucleotide compounds.
 32. A method of identifying one or morenucleic acid sequences amenable to antisense modulation, comprising thesteps of: (a) generating a library of nucleobase sequences in silicoaccording to defined criteria; (b) evaluating in silico a plurality ofvirtual oligonucleotides having the nucleobase sequences of (a)according to defined criteria; and (c) robotically assaying a pluralityof oligonucleotide compounds for one or more desired physical, chemicalor biological properties.
 33. A method of identifying one or morenucleic acid sequences amenable to antisense modulation, comprising thesteps of: (a) generating a library of nucleobase sequences in silicoaccording to defined criteria; (b) robotically synthesizing a pluralityof oligonucleotide compounds; and (c) robotically assaying a pluralityof oligonucleotide compounds for one or more desired physical, chemicalor biological properties.
 34. A method of identifying one or morenucleic acid sequences amenable to antisense modulation, comprising thesteps of: (a) evaluating in silico a plurality of virtualoligonucleotides according to defined criteria; (b) roboticallysynthesizing a plurality of oligonucleotide compounds; and (c)robotically assaying a plurality of oligonucleotide compounds for one ormore desired physical, chemical or biological properties.
 35. A methodof identifying one or more nucleic acid sequences amenable to antisensemodulation, comprising the steps of: (a) generating a library ofnucleobase sequences in silico according to defined criteria; (b)evaluating in silico a plurality of virtual oligonucleotides having thenucleobase sequences of (a) according to defined criteria; (c)robotically synthesizing a plurality of oligonucleotide compounds; and(d) robotically assaying a plurality of oligonucleotide compounds forone or more desired physical, chemical or biological properties.
 36. Amethod of identifying one or more nucleic acid sequences amenable toantisense modulation, comprising the steps of: (a) generating a libraryof nucleobase sequences in silico according to defined criteria; (b)choosing an oligonucleotide chemistry; (c) robotically synthesizing aset of oligonucleotide compounds having said nucleobase sequences ofstep (a) and said oligonucleotide chemistry of step (b); (d) roboticallyassaying said set of oligonucleotide compounds of step (c) for aphysical, chemical or biological activity; and (e) selecting a subset ofsaid set of oligonucleotide compounds of step (c) having a desired levelof physical, chemical or biological activity in order to generate saidset of compounds.
 37. A method of identifying one or more nucleic acidsequences amenable to antisense modulation, comprising the steps of: (a)generating a library of nucleobase sequences in silico according todefined criteria; (b) choosing an oligonucleotide chemistry; (c)evaluating in silico a plurality of virtual oligonucleotides having thenucleobase sequences of (a) according to defined criteria, and selectingthose having preferred characteristics, in order to generate a set ofpreferred nucleobase sequences; (d) robotically synthesizing a set ofoligonucleotide compounds having said preferred nucleobase sequences ofstep (b) and said oligonucleotide chemistry of step (c); (e) roboticallyassaying said set of oligonucleotide compounds of step (d) for aphysical, chemical or biological activity; and (f) selecting a subset ofsaid set of oligonucleotide compounds of step (d) having a desired levelof physical, chemical or biological activity in order to generate saidset of compounds.
 38. The method of claim 24, wherein said step ofrobotically assaying said plurality of nucleic acid sequences isperformed by computer-controlled real-time polymerase chain reaction orby computer-controlled enzyme-linked immunosorbent assay.
 39. The methodof claim 21, wherein said nucleic acid sequence is that of a genomicDNA, a cDNA, a product of a polymerase chain reaction, an expressedsequence tag, an mRNA or a structural RNA.
 40. The method of claim 21,wherein said nucleic acid sequence is a human nucleic acid.
 41. Acomputer formatted medium comprising computer readable instructions foridentifying active compounds.
 42. A computer formatted medium comprisingcomputer readable instructions for performing the method of any one ofclaims 1 to
 20. 43. A computer formatted medium comprising computerreadable instructions for performing a method of identifying one or morenucleic acid sequences amenable to antisense modulation.
 44. A computerformatted medium comprising computer readable instructions forperforming the method of any one of claims 21 to
 40. 45. A computerformatted medium comprising one or more nucleic acid sequences amenableto antisense modulation in computer readable form.
 46. A computerformatted medium comprising one or more nucleic acid sequences amenableto antisense modulation in computer readable form, wherein said one ormore nucleic acid sequences is identified according to the method of anyone of claims 21, 22 or 24-40.